The Development of an Isotope Dilution Mass Spectrometry Method for Interleukin-6 Quantification.

Inflammatory responses and tumor developments are closely related, with interleukin-6 (IL-6) playing important roles in both processes. IL-6 has been extensively identified as a potential tumor biomarker. This study developed an isotope dilution mass spectrometry (IDMS) method for quantifying IL-6 based on signature peptides. These peptides were screened by excluding those with missed cleavage or post-translational modification. The method's accuracy was verified using amino acid-based IDMS, in which purified IL-6 protein samples were quantified after hydrolyzing them into amino acids, and no significant difference was observed (p-value < 0.05). The method demonstrated good linearity and sensitivity upon testing. The specificity and matrix effect of the method were verified, and a precision study showed that the coefficient of variation was less than 5% for both the intra-day and inter-day tests. Compared to immunoassays, this method offers distinct advantages, such as the facilitation of multi-target analysis. Furthermore, the peptides used in this study are much more convenient for storage and operation than the antibodies or purified proteins typically used in immunoassays.

Gold nanostars for efficient in vitro and in vivo real-time SERS detection and drug delivery via plasmonic-tunable Raman/FTIR imaging.

The application of plasmonic-enhanced Raman imaging of cancer cells and drug delivery is gaining momentum. Here, we propose a new theranostic strategy based on an efficient plasmonic-tunable Raman/Fourier transform infrared (FTIR) spectroscopy imaging, to simultaneously evaluate the anticancer drug scattering cellular imaging and the Raman scattering molecular vibration signals in living cells. This technique allows to monitoring the drug release throughout the cell cycle and in vivo biodistribution and biocompatibility with low dose drug therapy (200 µg/mL) and low toxicity effect. This system can directly track in real-time the delivery and release of an anticancer drug (mitoxantrone, MTX) from gold nanostars in single living cells and in mice (healthy and lung cancer mice models), revealing a strong accumulation in the heart of healthy mice 5 min after administration and infiltration in the tumor site of lung cancer mice 5 h after systemic injection. This in vivo SERS detection method holds a great promise for application in image-guided cancer chemotherapy or as a nonspecific anti-inflammatory therapy for patients with cardiovascular diseases or chronic heart failure.

Bioresponsive antisense DNA gold nanobeacons as a hybrid in vivo theranostics platform for the inhibition of cancer cells and metastasis.

Gold nanobeacons can be used as a powerful tool for cancer theranostics. Here, we proposed a nanomaterial platform based on gold nanobeacons to detect, target and inhibit the expression of a mutant Kras gene in an in vivo murine gastric cancer model. The conjugation of fluorescently-labeled antisense DNA hairpin oligonucleotides to the surface of gold nanoparticles enables using their localized surface plasmon resonance properties to directly track the delivery to the primary gastric tumor and to lung metastatic sites. The fluorescently labeled nanobeacons reports on the interaction with the target as the fluorescent Cy3 signal is quenched by the gold nanoparticle and only emit light following conjugation to the Kras target owing to reorganization and opening of the nanobeacons, thus increasing the distance between the dye and the quencher. The systemic administration of the anti-Kras nanobeacons resulted in approximately 60% tumor size reduction and a 90% reduction in tumor vascularization. More important, the inhibition of the Kras gene expression in gastric tumors prevents the occurrence of metastasis to lung (80% reduction), increasing mice survival in more than 85%. Our developed platform can be easily adjusted to hybridize with any specific target and provide facile diagnosis and treatment for neoplastic diseases.

CD44v6 Monoclonal Antibody-Conjugated Gold Nanostars for Targeted Photoacoustic Imaging and Plasmonic Photothermal Therapy of Gastric Cancer Stem-like Cells.

Developing safe and effective nanoprobes for targeted imaging and selective therapy of gastric cancer stem cells (GCSCs) has become one of the most promising anticancer strategies. Herein, gold nanostars-based PEGylated multifunctional nanoprobes were prepared with conjugated CD44v6 monoclonal antibodies (CD44v6-GNS) as the targeting ligands. It was observed that the prepared nanoprobes had high affinity towards GCSC spheroid colonies and destroyed them completely with a low power density upon near-infrared (NIR) laser treatment (790 nm, 1.5 W/cm(2), 5 min) in vitro experiment. Orthotopic and subcutaneous xenografted nude mice models of human gastric cancer were established. Subsequently, biodistribution and photothermal therapeutic effects after being intravenously injected with the prepared nanoprobes were assessed. Photoacoustic imaging revealed that CD44v6-GNS nanoprobes could target the gastric cancer vascular system actively at 4 h post-injection, while the probes inhibited tumor growth remarkably upon NIR laser irradiation, and even extended survivability of the gastric cancer-bearing mice. The CD44v6-GNS nanoprobes exhibited great potential for applications of gastric cancer targeted imaging and photothermal therapy in the near future.

RNAi-based glyconanoparticles trigger apoptotic pathways for in vitro and in vivo enhanced cancer-cell killing.

Gold glyconanoparticles (GlycoNPs) are full of promise in areas like biomedicine, biotechnology and materials science due to their amazing physical, chemical and biological properties. Here, siRNA GlycoNPs (AuNP@PEG@Glucose@siRNA) in comparison with PEGylated GlycoNPs (AuNP@PEG@Glucose) were applied in vitro to a luciferase-CMT/167 adenocarcinoma cancer cell line and in vivo via intratracheal instillation directly into the lungs of B6 albino mice grafted with luciferase-CMT/167 adenocarcinoma cells. siRNA GlycoNPs but not PEGylated GlycoNPs induced the expression of pro-apoptotic proteins such as Fas/CD95 and caspases 3 and 9 in CMT/167 adenocarcinoma cells in a dose dependent manner, independent of the inflammatory response, evaluated by bronchoalveolar lavage cell counting. Moreover, in vivo pulmonary delivered siRNA GlycoNPs were capable of targeting c-Myc gene expression (a crucial regulator of cell proliferation and apoptosis) via in vivo RNAi in tumour tissue, leading to an ∼80% reduction in tumour size without associated inflammation.

Dual targeted immunotherapy via in vivo delivery of biohybrid RNAi-peptide nanoparticles to tumour-associated macrophages and cancer cells.

Lung cancer is associated with very poor prognosis and considered one of the leading causes of death worldwide. Here, we present highly potent and selective bio-hybrid RNAi-peptide nanoparticles that can induce specific and long-lasting gene therapy in inflammatory tumour associated macrophages (TAMs), via an immune modulation of the tumour milieu combined with tumour suppressor effects. Our data prove that passive gene silencing can be achieved in cancer cells using regular RNAi NPs. When combined with M2 peptide-based targeted immunotherapy that immuno-modulates TAMs cell-population, a synergistic effect and long-lived tumour eradication can be observed along with increased mice survival. Treatment with low doses of siRNA (ED50 0.0025-0.01 mg/kg) in a multi and long-term dosing system substantially reduced the recruitment of inflammatory TAMs in lung tumour tissue, reduced tumour size (∼95%) and increased animal survival (∼75%) in mice. Our results suggest that it is likely that the combination of silencing important genes in tumour cells and in their supporting immune cells in the tumour microenvironment, such as TAMs, will greatly improve cancer clinical outcomes.

A promising road with challenges: where are gold nanoparticles in translational research?

Nanoenabled technology holds great potential for health issues and biological research. Among the numerous inorganic nanoparticles that are available today, gold nanoparticles are fully developed as therapeutic and diagnostic agents both in vitro and in vivo due to their physicochemical properties. Owing to this, substantial work has been conducted in terms of developing biosensors for noninvasive and targeted tumor diagnosis and treatment. Some studies have even expanded into clinical trials. This article focuses on the fundamentals and synthesis of gold nanoparticles, as well as the latest, most promising applications in cancer research, such as molecular diagnostics, immunosensors, surface-enhanced Raman spectroscopy and bioimaging. Challenges to their further translational development are also discussed.

RGD-conjugated silica-coated gold nanorods on the surface of carbon nanotubes for targeted photoacoustic imaging of gastric cancer.

Herein, we reported for the first time that RGD-conjugated silica-coated gold nanorods on the surface of multiwalled carbon nanotubes were successfully used for targeted photoacoustic imaging of in vivo gastric cancer cells. A simple strategy was used to attach covalently silica-coated gold nanorods (sGNRs) onto the surface of multiwalled carbon nanotubes (MWNTs) to fabricate a hybrid nanostructure. The cross-linked reaction occurred through the combination of carboxyl groups on the MWNTs and the amino group on the surface of sGNRs modified with a silane coupling agent. RGD peptides were conjugated with the sGNR/MWNT nanostructure; resultant RGD-conjugated sGNR/MWNT probes were investigated for their influences on viability of MGC803 and GES-1 cells. The nude mice models loaded with gastric cancer cells were prepared, the RGD-conjugated sGNR/MWNT probes were injected into gastric cancer-bearing nude mice models via the tail vein, and the nude mice were observed by an optoacoustic imaging system. Results showed that RGD-conjugated sGNR/MWNT probes showed good water solubility and low cellular toxicity, could target in vivo gastric cancer cells, and obtained strong photoacoustic imaging in the nude model. RGD-conjugated sGNR/MWNT probes will own great potential in applications such as targeted photoacoustic imaging and photothermal therapy in the near future.

HAI-178 antibody-conjugated fluorescent magnetic nanoparticles for targeted imaging and simultaneous therapy of gastric cancer.

The successful development of safe and highly effective nanoprobes for targeted imaging and simultaneous therapy of in vivo gastric cancer is a great challenge. Herein we reported for the first time that anti-α-subunit of ATP synthase antibody, HAI-178 monoclonal antibody-conjugated fluorescent magnetic nanoparticles, was successfully used for targeted imaging and simultaneous therapy of in vivo gastric cancer. A total of 172 specimens of gastric cancer tissues were collected, and the expression of α-subunit of ATP synthase in gastric cancer tissues was investigated by immunohistochemistry method. Fluorescent magnetic nanoparticles were prepared and conjugated with HAI-178 monoclonal antibody, and the resultant HAI-178 antibody-conjugated fluorescent magnetic nanoparticles (HAI-178-FMNPs) were co-incubated with gastric cancer MGC803 cells and gastric mucous GES-1 cells. Gastric cancer-bearing nude mice models were established, were injected with prepared HAI-178-FMNPs via tail vein, and were imaged by magnetic resonance imaging and small animal fluorescent imaging system. The results showed that the α-subunit of ATP synthase exhibited high expression in 94.7% of the gastric cancer tissues. The prepared HAI-178-FMNPs could target actively MGC803 cells, realized fluorescent imaging and magnetic resonance imaging of in vivo gastric cancer, and actively inhibited growth of gastric cancer cells. In conclusion, HAI-178 antibody-conjugated fluorescent magnetic nanoparticles have a great potential in applications such as targeted imaging and simultaneous therapy of in vivo early gastric cancer cells in the near future.

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.

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 αvß3 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.

The immunotoxicity of graphene oxides and the effect of PVP-coating.

Graphene oxide (GO) immunotoxicity is not clarified well up to date. Herein we reported the effects of GOs with and without polyvinylpyrrolidone (PVP) coating on human immune cells such as dendritic cells (DCs), T lymphocytes and macrophages. Human immune cells such as dendritic cells (DCs), T lymphocytes and macrophages were isolated from health donated bloods, PVP-coating GO (PVP-GO) exhibited lower immunogenicity compared with pure GO on the aspect of inducing differentiation and maturation of dendritic cells (DCs), the levels of secreted TNF-α and IL-1ß had no obvious difference between two groups, yet the secretion of IL-6 remained in PVP-coating GO group. In addition, PVP-coating GO delayed significantly the apoptotic process of T lymphocytes, at the same time, and exhibited anti-phagocytosis ability against macrophages and markedly enhanced the physiological activity of macrophages. In conclusion, PVP-coating GO possesses good immunological biocompatibility and immunoenhancement effects in vitro, and is likely to be an available candidate of immunoadjuvant in the future.

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.

DiR-labeled Embryonic Stem Cells for Targeted Imaging of in vivo Gastric Cancer Cells.

Embryonic stem (ES) cells have great potential in applications such as disease modeling, pharmacological screening and stem cell therapies. Up to date, there is no related report on the use of ES cells as tracking and contrast reagents of cancer cells in vivo. Herein we report that DiR-labeled murine ES cells can recognize and target gastric cancer cells in vivo. DiR-labeled murine ES (mES) cells (5×10(6)) were intravenously injected into gastric tumor-bearing mice. The biodistribution of DiR-labeled mES cells was monitored by IVIS imaging within 24 h. Major organs were harvested and analyzed by immunofluorescence staining and Western blotting. Chemotaxis assay was employed to investigate the chemotaxis of ES cells tracking cancer cells. Fluorescent imaging results showed that DiR-labeled mES cells targeted gastric cancer tissue in vivo as early as 10 min post-injection, reaching a peak at 2h post-injection. Immunofluorescence staining and Western blotting results showed gastric cancer tissues specifically expressed SSEA-1. In vitro migration tests confirmed that mES cells actively moved to test sites with different concentration of CXCL12 in a dose-dependent manner. In conclusion, DiR-labeled mES cells may be used for gastric cancer targeted imaging in vivo, and have great potential in applications such as identifying and imaging of early gastric cancer in near future.

HER2 monoclonal antibody conjugated RNase-A-associated CdTe quantum dots for targeted imaging and therapy of gastric cancer.

Successful development of safe and effective nanoprobes for targeted imaging and selective therapy of in-situ gastric cancer is a great challenge. Herein, one kind of multifunctional HER2 monoclonal antibody conjugated RNase A-associated CdTe quantum dot cluster (HER2-RQDs) nanoprobes was prepared, its cytotoxicity was evaluated. Subcutaneous gastric cancer nude mouse models and in-situ gastric cancer SCID mouse models were established, and were intravenously injected HER2-RQDs nanoprobes, the bio-distribution and therapeutic effects of HER2-RQDs in vivo were evaluated. Results showed that HER2-RQDs nanoprobes could selectively kill gastric cancer MGC803 cells, could target imaging subcutaneous gastric cancer cells at 3 h post-injection, and in-situ gastric cancer cells at 6 h post-injection, and could inhibit the growth of gastric cancer tissues and extended survival time of gastric cancer bearing mouse models, which is closely associated with destroying functional RNAs in cytoplasm by RNase A released from HER2-RQDs nanoprobes, preventing protein synthesis and inducing cell apoptosis. High-performance HER2-RQDs nanoprobes exhibit great potential in applications such as in-situ gastric cancer targeted imaging, and selective therapy in the near future.

Development of Polysorbate 80/Phospholipid mixed micellar formation for docetaxel and assessment of its in vivo distribution in animal models.

Docetaxel (DTX) is a very important member of taxoid family. Despite several alternative delivery systems reported recently, DTX formulated by Polysorbate 80 and alcohol (Taxotere®) is still the most frequent administration in clinical practice. In this study, we incorporated DTX into Polysorbate 80/Phospholipid mixed micelles and compared its structural characteristics, pharmacokinetics, biodistribution, and blood compatibility with its conventional counterparts. Results showed that the mixed micelles loaded DTX possessed a mean size of approximately 13 nm with narrow size distribution and a rod-like micelle shape. In the pharmacokinetics assessment, there was no significant difference between the two preparations (P > 0.05), which demonstrated that the DTX in the two preparations may share a similar pharmacokinetic process. However, the Polysorbate 80/Phospholipid mixed micelles can increase the drug residence amount of DTX in kidney, spleen, ovary and uterus, heart, and liver. The blood compatibility assessment study revealed that the mixed micelles were safe for intravenous injection. In conclusion, Polysorbate 80/Phospholipid mixed micelle is safe, can improve the tumor therapeutic effects of DTX in the chosen organs, and may be a potential alternative dosage form for clinical intravenous administration of DTX.

BRCAA1 monoclonal antibody conjugated fluorescent magnetic nanoparticles for in vivo targeted magnetofluorescent imaging of gastric cancer.

BACKGROUND: Gastric cancer is 2th most common cancer in China, and is still the second most common cause of cancer-related death in the world. How to recognize early gastric cancer cells is still a great challenge for early diagnosis and therapy of patients with gastric cancer. This study is aimed to develop one kind of multifunctional nanoprobes for in vivo targeted magnetofluorescent imaging of gastric cancer. METHODS: BRCAA1 monoclonal antibody was prepared, was used as first antibody to stain 50 pairs of specimens of gastric cancer and control normal gastric mucous tissues, and conjugated with fluorescent magnetic nanoparticles with 50 nm in diameter, the resultant BRCAA1-conjugated fluorescent magnetic nanoprobes were characterized by transmission electron microscopy and photoluminescence spectrometry, as-prepared nanoprobes were incubated with gastric cancer MGC803 cells, and were injected into mice model loaded with gastric cancer of 5 mm in diameter via tail vein, and then were imaged by fluorescence optical imaging and magnetic resonance imaging, their biodistribution was investigated. The tissue slices were observed by fluorescent microscopy, and the important organs such as heart, lung, kidney, brain and liver were analyzed by hematoxylin and eosin (HE) stain method. RESULTS: BRCAA1 monoclonal antibody was successfully prepared, BRCAA1 protein exhibited over-expression in 64% gastric cancer tissues, no expression in control normal gastric mucous tissues, there exists statistical difference between two groups (P < 0.01). The BRCAA1-conjugated fluorescent magnetic nanoprobes exhibit very low-toxicity, lower magnetic intensity and lower fluorescent intensity with peak-blue-shift than pure FMNPs, could be endocytosed by gastric cancer MGC803 cells, could target in vivo gastric cancer tissues loaded by mice, and could be used to image gastric cancer tissues by fluorescent imaging and magnetic resonance imaging, and mainly distributed in local gastric cancer tissues within 12 h post-injection. HE stain analysis showed that no obvious damages were observed in important organs. CONCLUSIONS: The high-performance BRCAA1 monoclonal antibody-conjugated fluorescent magnetic nanoparticles can target in vivo gastric cancer cells, can be used for simultaneous magnetofluorescent imaging, and may have great potential in applications such as dual-model imaging and local thermal therapy of early gastric cancer in near future.

A prototype of giant magnetoimpedance-based biosensing system for targeted detection of gastric cancer cells.

A targeted detection of gastric cancer cells is achieved by combining the giant magnetoimpedance (GMI)-based biosensing system and RGD-4C peptide coupled, chitosan covered superparamagnetic iron oxide particles (RGD-Fe(3)O(4)@chitosan). The micro-patterned GMI sensor for targeted detection is made of Co-based ribbon and fabricated by micro electromechanical system (MEMS) technology. Functionalized nanoparticles were designed by coating Fe(3)O(4) with chitosan and conjugating with RGD-4C peptides. The targeted cells were trickled down into the detection area of the system. The detection of each sample is carried out in ten-fold manner and average value is taken as the final result. This system can identify the differences between targeted cells and non-targeted cells. It is of considerable interest due to its potential application in the biomedical field of various specific detections.

The potential of magnetic nanocluster and dual-functional protein-based strategy for noninvasive detection of HBV surface antibodies.

Magnetic nanoclusters (MNCs) were synthesized in a one-pot process, carboxylic MNCs and dual-functional protein were prepared and used to capture hepatitis B virus surface antibodies (anti-HBs) in simulated diseased oral mucosal transudate (OMT) samples. The specific substrate of dual-functional protein, dual-labeled double-chained DNA molecules, based on Fluorescence Resonance Energy Transfer (FRET), was used to amplify the detection signal and the detection limit of 0.1 ng mL(-1) of anti-HBs monoclonal antibodies was achieved. Combination MNCs with dual-functional protein enables the noninvasive detection of hepatitis B virus (HBV) surface antibodies in OMT samples, showing promise as a diagnostic tool for the OMT diagnosis of infectious diseases with sensitive, specific and facile capabilities.

Preparation of HIV-1 Env protein and establishment of ultrasensitive detection method of HIV-1 gp41 antibody.

Preparation of HIV-1 Env protein and development of ultrasensitive detection method of HIV-1 gp41 antibody are challengeable tasks for early diagnosis of patients with HIV virus. The env gene fragments were obtained from the plasmid with HIV-1 env by PCR, and were cloned into T vector, and then were sequenced. The linear env gene fragments were prepared by Rapid Translation System (RTS) Linear Template Kit, the Env proteins were obtained by RTS Kit, and were purified by magnetic bead method. The Env protein's bioactivity was identified by Western Blotting. The prepared Env proteins were labeled with CdTe quantum dots (QDs), and the goat anti-HIV-1 gp41 IgG antibodies were detected by CdTe QDs-labeled Env proteins, the fluorescent signals were recorded by photoluminescent spectroscopy. Results show that the prepared Env proteins own bioactivity, the CdTe QDs-labeled Env proteins can combine with anti-HIV-1 gp41 IgG antibodies, and formed the Env-gp41-CdTe QDs complex, the fluorescent intensity of final products were negatively associated with the concentration of anti-HIV-1 gp41 IgG antibodies in sample, the limitation of detection is 100 pg/mL. In conclusion, HIV-1 Env proteins with bioactivity were successfully prepared by RTS, established CdTe QDs labeled Env proteins-based fluorescent immunoassay can successfully detect HIV-1 gp41 IgG antibodies, which has great potential in early diagnosis of HIV.