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Sweet as sugar, hard as carbon: A hierarchical core-shell 3D graphene network biosensor for glucose detection

A biosensor is a device that uses biological molecules, typically enzymes, to specifically detect the presence of a chemical or a metabolic intermediate (referred to as the analyte) in a diagnostic setting. A biosensor acts as the platform upon which a biochemical reaction, initiated by the analyte, is converted to an electric current that is accurately quantified during a subsequent step. Biosensors have wide clinical applicability. For instance, the detection of blood sugar, which is among the most frequently measured physiological variable, is achieved with biosensors.

Recent years have seen rapid advancements in the use of nanoparticles, nanowires and nanotubes as biosensor platforms. These innovative nanostructures are electrochemically active, chemically stable, have large surface areas and are biocompatible – all of which are desirable attributes for developing biosensors. Of note is the observation that graphene, a substance known for its high electrical conductivity,  lends itself to biosensor development due to its relative ease of manufacture together with its ability to form composites with other electrochemically active nanostructures.

Early prototypes of graphene-based biosensors were inefficient for two main reasons. First, the clumping of graphene sheets reduced the accessible surface area. As a consequence, the biosensor/analyte interface was greatly reduced. Second, the restacking of graphene sheets introduced electrical resistance due to intersheet contacts. To overcome these hurdles, a research group led by Azam Iraji Zad at the Institute for Nanoscience and Nanotechnology (INST), Tehran, Iran developed a freestanding, porous 3D graphene network (3DGN) which was further modified with metal oxide nanostructures as a platform upon which an enzymatic reaction could occur.

This proof-of-concept study uses the glucose oxidase enzyme for the rapid and selective detection of glucose. The 3DGN, a graphene skeleton with multiple pores, is the core of the nanostructure. Atop the 3DGN, the researchers first grew uniformly spaced ZnO nanorods, which served to hold the enzyme in place. In a subsequent step, MnO2, known to be biocompatible and stable, was deposited onto the ZnO nanorods, thus forming a multilayered hierarchical structure with an average diameter of 100nm. The researchers propose that that the complex architecture of the nanostructure serves to facilitate the electron transfer process, which is the fundamental biochemical mechanism driving the enzymatic reaction.

In principle, the inner parts of the ZnO nanotubes increase the accessible surface area of the nanostructure and enhance the biosensor/analyte interface. In theory, the 3DGN biosensor is expected to respond quicker and have improved sensitivity when compared to other enzyme-based glucose detection devices. The study tested the 3DGN biosensor using a method called amperometry which is used routinely in research laboratories to detect ions – the byproduct of enzymatic reactions. The study found that the 3DGN biosensors had a response time of less than 3 seconds; a value indicative of a competitive advantage over other enzyme-based glucose biosensors. Intriguingly, the study also found that the 3DGN was very sensitive and could detect extremely low concentrations (10nM) of glucose.

The study strongly suggests that 3DGN biosensors could be used as an accurate sensing platform for chemicals and biomolecules. The findings further support the argument that composite nanostructures with complex architecture could find applicability in human health and beyond.

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Elham Asadian, Saeed Shahrokhian and Azam Iraji Zadac
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Now you see me: autofluorescent nanoparticles for live cell imaging and biodegradation modeling

There is an increasing need for novel technologies to facilitate in vivo tissue visualization and drug delivery. However, this need is largely unmet due to the challenges associated with creating biocompatible materials that meet safety standards. In addition, the potential health risks associated with the accumulation of non-degradable imaging agents and drug carries represents a major obstacle in the innovation pipeline.

The intrinsic autofluorescent, biodegradable and biocompatible properties of Bovine Serum Albumin (BSA) is well appreciated. However, BSA has short excitation and emission wavelengths, which substantially restricts any in vivo biomedical applications.  Motivated by a recent report suggesting that glutaraldehyde (GA)-crosslinking induces autofluorescence in protein-based nanoparticles by modifying a series of C=C and C=N bonds, a team led by Yu Lei at the Department of Biomedical Engineering, University of Connecticut, developed low-cost, non-toxic, BSA-based protein nanoparticles (average size ~40 nm) for live cell imaging and biodegradation analysis.

The nanoparticles were generated by adding drops of a prepared BSA solution to glutaraldehyde/n-butanol solution at high-speed, and the resulting product heated at 121°C to ensure sterility. Interestingly, a similar reaction carried out in the absence of the GA crosslinker did not produce autofluorescent BSA nanoparticles, suggesting that GA was indeed playing an important role in chemically transforming BSA. Using UV-visible spectroscopy, the investigators observed that BSA nanoparticles exhibited strong autofluorescence at both green (530 nm) and red (630 nm) wavelengths.

The BSA nanoparticles were not uniform in structure, owing to the random points of crosslinking within BSA, and also due to the ensuing condensation reaction that occurs during the sterilization step. Therefore, a clear mechanistic explanation for the strong autofluorescence warrants further investigation. However, the investigators speculate that GA-crosslinking and heating could result in new C=N bonds, which could synergize with the C=C bonds from tryptophan, tyrosine, phenylalanine and histidine residues with BSA, leading to enhanced green and red fluorescence.

The team went on to demonstrate the utility of the BSA nanoparticles in biomedical applications such as imaging and biodegradation. They used fluorescent microscopy techniques to visualize the entry of BSA nanoparticles into human kidney cells grown in vitro. The study also found that the BSA nanoparticles were completely degraded within 18 days of injection in mice. A mathematical model for the distribution and biodegradation of the nanoparticles was in good agreement with the experimental results. Finally, to add an additional line of evidence supporting the biocompatible nature of the BSA nanoparticles, the investigators looked for signs of tissue damage in the region surrounding the site of injection, together with an analysis of internal organs including the pancreas, liver and kidney, and report that the BSA nanoparticles are biocompatible.

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Xiaoyu Ma, Derek Hargrove, Qiuchen Dong, Donghui Song, Jun Chen, Shiyao Wang, Xiuling Lu, Yong Ku Cho, Tai-Hsi Fan and  Yu Lei
DOI: 10.1039/c6ra06783b
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The heat is on: cancer-drug loaded nanoparticles for photothermal therapy

Photothermal therapy is an emerging area of cancer treatment. Here, a photothermal agents, often nanoparticles (NPs) with a resonance peak in the 700-1200nm range, are delivered to the tumor site and are subsequently activated by light in the Near Infrared (NIR) range. As a consequence, tumor cells are thermally ablated.

In a study led by Xiaolin Li and colleagues at the Key Laboratory for Thin Film and Microfabrication and Changzheng Hospital in China, scientists used SiO2@Au core-shell NPs chemically conjugated via PEGylation to graphene oxide (GO) in conjunction with a chemotherapeutic agent to target prostate cancer cells in vitro. Using the chemotherapeutic agent Docetaxel (Dtxl),  which is among the leading front line treatments for patients diagnosed with prostate cancer, the team demonstrated that Dtxl-loaded SiO2@Au@GO NPs, when activated with light in the NIR range, significantly curbed the survival of DU145 prostate cancer cells.

While SiO2@Au core-shell NPs have been used previously by other research groups to study their ability to remove tumors, Li’s team fabricated SiO2@Au@GO NPs to take advantage of their relatively low cost, large specific surface area, and efficient loading and delivery of water-soluble aromatic drug molecules. This one-two punch strategy was realized via a double shell, multifunctional approach: the inner core SiO2@Au NPs served as a photothermal inducer to bring about cellular cytotoxicity; the outer GO NPs carried the antitumor drug, Dtxl. The study found that exposing DU145 cells to the NPs alone for 24h did not result in overt cell death, suggesting that the NPs have a good safety profile. Importantly, the study showed that when NP-treated cell cultures were irradiated with a 780nm NIR laser, there was a significant decline in viable cells over a 24h period.

The study demonstrates that Dtxl-loaded SiO2@Au@GO NPs could be manufactured and potentially used an an antitumor agent for the treatment of prostate cancer. Moreover, these findings illuminate the untapped potential of NP-based photothermal agents as adjuvant agents in oncology clinical trials in the near future.

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Xiaolin Li,   Zhi Yang,   Nantao Hu,   Liying Zhang,   Yafei Zhang and   Lei Yin
DOI: 10.1039/C6RA03886G
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Modelling lung cancer: tumor cells on collagen scaffolds

Non-small-cell lung cancer (NSCLC) is among the leading causes of cancer-related deaths globally. Our understanding of the way tumors grow, spread and respond to therapy is driven largely by studies conducted on tumor cells growing as monolayers in plastic cell culture flasks in laboratories across the world. The ability to develop novel and more effective cancer-fighting drugs is dependent, in part, on developing cell culture systems that allow scientists to better observe how tumor cells grow in a three dimensional, physiologically relevant environment.

SEM images of the collagen meshwork and A549 cell aggregates (noted by the arrow head) formed during the
3D cultivation in vitro.

The tumor microenvironment (TM) is the area that immediately surrounds a tumor and includes non-cancer cells together with secreted proteins called the extracellular matrix (ECM), which supports tumor growth. Monolayer cell cultures, although utilized widely, cannot accurately mimic the TM. For instance, cell-cell and cell-ECM interactions that influence tumor growth cannot be observed in great detail with conventional monolayer cultures. Inspired by the up-and-coming field of tumor engineering, which aims to construct culture models that recapitulate aspects of the TM, a team of researchers led by Dr. Dan-Dan Wang at the Chinese Academy of Sciences developed a 3D culture system wherein A549 cells (immortal lung cancer cells of human origin) grow on a collagen hydrogel scaffold.

To demonstrate the utility of the 3D culture system, the study measured cell viability and showed that cells in the collagen hydrogel scaffold were alive for extended periods (>12 days) in vitro. The study also assessed the appearance of artificial A549 tumors growing on the hydrogel to demonstrate that 3D cultures more closely recapitulate the morphology of tumors growing within human tissues.

The proliferation of A549 cells is driven by the activation of a cell surface protein called Epidermal Growth Factor Receptor (EGFR), which in turn switches on genes that sustain cell growth and cell division. The team observed that Gefitinib, a drug known to disrupt growth-promoting signals arising at EGFR, was able to significantly constrain A549 cell proliferation in 3D cultures. Interestingly, the team reports that a higher concentration of Gefitinib was required to curb cell growth in 3D cultures compared to monolayers due to the complex architecture of the artificial tumors in 3D cultures.

Collectively, this study demonstrates an improved culture model of human lung cancer. Since collagen is an important component of the ECM, the study sets the stage for future efforts to better recapitulate the TM in vitro. The collagen hydrogel scaffold system could serve as in important tool in the discovery of targeted therapies for lung cancer.

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Dan-Dan Wang,   Wei Liu,   Jing-Jie Chang,   Xu Cheng,   Xiu-Zhen Zhang,   Hong Xu,   Di Feng,   Li-Jun Yu and   Xiu-Li Wang
RSC Adv., 2016, 6, 24083-24090
DOI: 10.1039/C6RA00229C
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Hitching a ride: recombinant DNA delivery into mammalian cells via nanoparticle-based vehicles

Transfection is the process of introducing genetic material, typically DNA, into mammalian cells. This technique has proven indispensable in understanding signaling networks that govern cellular function. To better understand the function of a given protein, molecular biologists routinely transfect cells with DNA (i.e. recombinant DNA). This enters cells in culture and subsequently encodes the specific protein under study. The recombinant DNA is combined with a transfection reagent, typically Lipofectamine, to facilitate its entry into cells.

A study conducted by Neuhaus and colleagues, at the Inorganic Chemistry and Center for Nanointegration (CeNIDE) in Germany, utilizes calcium phosphate nanoparticles (CPNPs) as vehicles to deliver recombinant DNA into cells. CPNPs have previously been shown to spontaneously bind DNA, thus supporting the notion that they could be used as transfection agents. The approach requires that CPNPs first be mixed with a buffer containing recombinant DNA before being added to cultures containing actively growing mammalian cells.

Despite its simplistic approach, the transfection process in general has a few technical limitations. First, not all cells in culture uptake the recombinant DNA. This leads to reduced transfection efficiency. Second, the transfection efficiency is strongly influenced by the cell type (i.e. distinct cell forms within a species). And third, cells interpret recombinant DNA as ‘foreign’ genetic material and trigger alarms which culminate in cell death.

Images demonstrating the uptake of green flourescent nanoparticles by different cell types

To better assess the utility of CPNPs as transfection agents, the study’s authors first transfected ten different cell types with DNA. The DNA in their study encoded a protein that fluoresces green when excited at a specific wavelength. Using Lipofectamine as a comparator reagent, the study assessed the transfection efficiency of CPNPs by measuring the proportion of cells that glowed green under a fluorescent microscope. The study also highlighted the differences in transfection efficiencies between different cell types. The authors propose that CPNPs represent promising candidates as transfection agents and therefore warrant further study.

Clinical trials utilizing nucleotide-based targeted therapies for multiple human diseases are on the rise. CPNPs may represent the new breed of nucleotide-based drug delivery agents in the years to come.

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Nanoparticles as transfection reagents: a comprehensive study with ten different cell lines
Bernhard Neuhaus,  Benjamin Tosun, Olga Rotan, Annika Frede, Astrid M. Westendorf and Matthias Epple
RSC Adv., 2016,6, 18102-18112
DOI: 10.1039/C5RA25333K

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