News & Views
Research Highlights Highlights from the latest articles in nanomedicine
Application of spirothiopyran to generate light-induced self-assembly of metal nanoparticles Evaluation of: Shiraishi Y, Tanaka K, Shirakawa E et al. Light-triggered self-assembly of gold nanoparticles based on photoisomerization of spirothiopyran. Angew. Chem. Int. Ed. 52(32), 8304–8308 (2013). Gold nanoparticles (AuNPs) offer tremendous potential for cancer diagnosis and therapy owing to the variety of unique chemical and electronic properties. These properties heavily depend on the size of the AuNPs, which makes the accuracy of size control vital for their advanced processing. The light-triggered self-assembly of AuNPs appears to be the most effective method for size control because it is noninvasive and can be selectively delivered to a precise location. In this study, Shiraishi and coworkers developed a new method to promote light-triggered self-assembly of AuNPs. Rather than covalently attaching photoreactive molecules to the surfaces of AuNPs, the group placed AuNPs in a
solution with spirothiopyran, a dye that photoisomerizes under UV irradiation. The original form does not interact with AuNPs; however, the isomerized form binds to the surface of the AuNP and alters its surface charge, thus triggering aggregation of the particles. The group used transmission electron microscopy to confirm that the AuNPs aggregated when placed in solution with spirothiopyran and subjected to UV irradiation. These data, along with dynamic laser scattering analysis, suggest that the size of the aggregate AuNPs depends primarily on irradiation time, and that the standard deviation of aggregate sizes for each time interval is less than 25%. They also found that extinguishing the UV light completely suppresses any increase in aggregation, which implies that cycling the UV light on and off can be used to precisely control aggregation. Ultimately, this study describes a novel technique that may contribute to improvements in the assembly efficiency and processing of metal nanoparticles, particularly AuNPs or other metal nanoparticles.
Hannah Friedman1, Alex T Holt1 & Wellington Pham*1 Institute of Imaging Science, Vanderbilt University, 1161 21st Avenue South, Nashville, TN 37232, USA *Author for correspondence: Tel.: +1 615 936 7621 Fax: +1 615 322 0734 [email protected] 1
Financial & competing interests disclosure This work is supported by a grant from the NIH, NCI R01CA160700. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript.
Using novel fluorescent decay kinetics near-infrared dyes encased in nanoparticles for imaging Evaluation of: Hoffmann K, Behnke T, Drescher D, Kneipp J, Resch-Genger U. Near-infraredemitting nanoparticles for lifetimebased multiplexed analysis and imaging of living cells. ACS Nano 7(8), 6674–6684 (2013).
10.2217/NNM.13.174 © 2013 Future Medicine Ltd
Bioanalysis and biomedical imaging commonly employ fluorescent labels. However, interest has peaked recently on expanding the use of fluorescence techniques to investigate the interactions of the components of a given biological system. One approach utilizes spectral multiplexing dyes, which are excitable at the same wavelength but Nanomedicine (2013) 8(12), 1909–1911
part of
ISSN 1743-5889
1909
Research Highlights News & Views – News
distinguishable by different emission spectra. However, overlapping absorption and emission spectra, as well as small Stokes shifts (the difference in the maxima of the absorption and emission spectra), hamper this method. The novel approach used by Hoffmann and associates applied the concept of fluorescence lifetime multiplexing in which fluorophores are distinguished based on fluorescent decay kinetics. The
fluorophores involved must have different lifetimes but be excitable at the same wavelength and detectable in the same spectral window. Fitting a mathematical model of the decay kinetics allows the determination of the ratio of the components. To implement this approach, the investigators used two dye-loaded polystyrene nano particles: Itrybe, an asymmetrical cyanide; and Sq730, a squaraine dye. Itrybe has a large Stokes shift, while Sq730 has narrow and structured absorption bands. Notably, both have significantly different decay kinetics, where dye encapsulation via polystyrene nanoparticles enables the
reporter decay kinetics to become independent of the probe’s environment. In proof-of-concept studies using fluorescent imaging of live mouse 3T3 fibroblast cells and J774 macrophages, the investigators demonstrate the potential of fluorescence lifetime-based analysis to spatially distinguish the location of different sized particles within the cells. By deconvolving the decay kinetics, they also quantitatively determined the differential particle size uptake. These results demonstrate the potential of nanoparticle-based fluorescence lifetime multiplexing in cellular studies and cell-based assays.
Low-frequency fluctuations of an atomic force microscope cantilever is capable of monitoring nanometer-scale activity of bacteria in real time Evaluation of: Longo G, Alonso-Sarduy L, Marques Rio L et al. Rapid detection of bacterial resistance to antibiotics using AFM cantilevers as nanomechanical sensors. Nat. Nanotechnol. 8(7), 522–526 (2013). The widespread use of drugs has increased the number of multidrug-resistant bacteria. Therefore, in the overall approach to managing infection, there is a critical need to develop tools that can rapidly characterize bacterial response to antibiotics. Although techniques for the
detection and characterization of bacterial resistance to antibiotics are available, the process is time consuming. In this study, Longo and coworkers demonstrated that the fluctuations of highly sensitive atomic force microscope cantilevers can be used to quickly detect quantitatively low concentrations of bacteria according to their metabolism and response to antibiotic treatment. The measurement is achieved by immobilizing live bacteria on a canti lever sensor. The movements of the bacteria cause an increase in the amplitude of the fluctuations of the sensor, which can be monitored using an atomic force microscope. The authors tested this technology
on Escherichia coli and Staphylococcus aureus and confirmed its reliability for reporting bacterial metabolism. Before the bacteria were introduced, the fluctuations were very slight; however, the fluctuations increased and appeared to become strongly dependent on bacterial activity after their introduction. Following treatment with ampicillin, the fluctuations diminished to very low values and remained as such after the antibiotic was flushed out. This suggests that the ampicillin eliminated the bacteria. In general, the application of this novel technique is not limited to bacteria, as it can be used for studying cells or protein conformational changes.
Lipid nanoparticles as ideal delivery modules for siRNA Evaluation of: Gilleron J, Querbes W, Zeigerer A et al. Image-based analysis of lipid nanoparticle‑mediated siRNA delivery, intracellular trafficking and endosomal escape. Nat. Biotechnol. 31(7), 638–646 (2013). 1910
Synthetic siRNAs have emerged as a promising therapeutic modality to silence disease-associated genes. However, while the delivery of an active form of siRNA is a complex process, it is critical in the successful development of siRNA-based drugs currently being tested in several clinical trials. Liposomes and polycationic Nanomedicine (2013) 8(12)
polymers have been used for siRNA delivery, although those techniques suffer from various levels of efficiency and toxicity, thus making them clinically unsatisfactory. By contrast, lipid nanoparticles (LNPs) are the most advanced delivery systems for siRNAs. The construct is comprised of multiple lipids that self-assemble future science group
Research Highlights News – into 60–80‑nm particles, thereby encapsulating the siRNA molec ules. Nevertheless, the underlying mechanism of LNP-mediated siRNA delivery is not yet fully understood. In this study, Gilleron et al. reported that LNPs enter cells via both constitutive and inducible pathways in a cell type-specific manner using clathrin-mediated endoc ytosis (CME) as well as macropinoc ytosis. Inhibition of CME using dynasore (a small-molecule inhibitor of dynamin), a GTPase required for membrane fission in CME or macropinocytosis using ethylisopropylamiloride reduced uptake by 50–70%,
future science group
which suggests that both pathways are required. Furthermore, the authors used time-lapse confocal fluorescence micro scopy to examine the release of siRNA into the cytosol. The results demonstrated that siRNAs delivered by LNPs are not massively released owing to the bursting of individual endosomes or the permeabilization of the limiting membrane of endosomal compartments. It is likely that such release is restricted to a limited number of siRNAs and is, therefore, undetectable using standard fluorescence microscopy methods. However, by using electron microscopy to study the release
www.futuremedicine.com
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using LNP–siRNA–gold, the authors discovered that siRNA–gold escaped from endosomes in hepatocytes in vivo and reached a level of 186.05 siRNA–gold per cell after 6 h. In general, the results reported in this work add insight to LNPmediated siRNA delivery, which may help guide the development of the next generation of delivery systems for RNAi therapeutics.
1911
Research Highlights Highlights from the latest articles in nanomedicine
Application of spirothiopyran to generate light-induced self-assembly of metal nanoparticles Evaluation of: Shiraishi Y, Tanaka K, Shirakawa E et al. Light-triggered self-assembly of gold nanoparticles based on photoisomerization of spirothiopyran. Angew. Chem. Int. Ed. 52(32), 8304–8308 (2013). Gold nanoparticles (AuNPs) offer tremendous potential for cancer diagnosis and therapy owing to the variety of unique chemical and electronic properties. These properties heavily depend on the size of the AuNPs, which makes the accuracy of size control vital for their advanced processing. The light-triggered self-assembly of AuNPs appears to be the most effective method for size control because it is noninvasive and can be selectively delivered to a precise location. In this study, Shiraishi and coworkers developed a new method to promote light-triggered self-assembly of AuNPs. Rather than covalently attaching photoreactive molecules to the surfaces of AuNPs, the group placed AuNPs in a
solution with spirothiopyran, a dye that photoisomerizes under UV irradiation. The original form does not interact with AuNPs; however, the isomerized form binds to the surface of the AuNP and alters its surface charge, thus triggering aggregation of the particles. The group used transmission electron microscopy to confirm that the AuNPs aggregated when placed in solution with spirothiopyran and subjected to UV irradiation. These data, along with dynamic laser scattering analysis, suggest that the size of the aggregate AuNPs depends primarily on irradiation time, and that the standard deviation of aggregate sizes for each time interval is less than 25%. They also found that extinguishing the UV light completely suppresses any increase in aggregation, which implies that cycling the UV light on and off can be used to precisely control aggregation. Ultimately, this study describes a novel technique that may contribute to improvements in the assembly efficiency and processing of metal nanoparticles, particularly AuNPs or other metal nanoparticles.
Hannah Friedman1, Alex T Holt1 & Wellington Pham*1 Institute of Imaging Science, Vanderbilt University, 1161 21st Avenue South, Nashville, TN 37232, USA *Author for correspondence: Tel.: +1 615 936 7621 Fax: +1 615 322 0734 [email protected] 1
Financial & competing interests disclosure This work is supported by a grant from the NIH, NCI R01CA160700. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript.
Using novel fluorescent decay kinetics near-infrared dyes encased in nanoparticles for imaging Evaluation of: Hoffmann K, Behnke T, Drescher D, Kneipp J, Resch-Genger U. Near-infraredemitting nanoparticles for lifetimebased multiplexed analysis and imaging of living cells. ACS Nano 7(8), 6674–6684 (2013).
10.2217/NNM.13.174 © 2013 Future Medicine Ltd
Bioanalysis and biomedical imaging commonly employ fluorescent labels. However, interest has peaked recently on expanding the use of fluorescence techniques to investigate the interactions of the components of a given biological system. One approach utilizes spectral multiplexing dyes, which are excitable at the same wavelength but Nanomedicine (2013) 8(12), 1909–1911
part of
ISSN 1743-5889
1909
Research Highlights News & Views – News
distinguishable by different emission spectra. However, overlapping absorption and emission spectra, as well as small Stokes shifts (the difference in the maxima of the absorption and emission spectra), hamper this method. The novel approach used by Hoffmann and associates applied the concept of fluorescence lifetime multiplexing in which fluorophores are distinguished based on fluorescent decay kinetics. The
fluorophores involved must have different lifetimes but be excitable at the same wavelength and detectable in the same spectral window. Fitting a mathematical model of the decay kinetics allows the determination of the ratio of the components. To implement this approach, the investigators used two dye-loaded polystyrene nano particles: Itrybe, an asymmetrical cyanide; and Sq730, a squaraine dye. Itrybe has a large Stokes shift, while Sq730 has narrow and structured absorption bands. Notably, both have significantly different decay kinetics, where dye encapsulation via polystyrene nanoparticles enables the
reporter decay kinetics to become independent of the probe’s environment. In proof-of-concept studies using fluorescent imaging of live mouse 3T3 fibroblast cells and J774 macrophages, the investigators demonstrate the potential of fluorescence lifetime-based analysis to spatially distinguish the location of different sized particles within the cells. By deconvolving the decay kinetics, they also quantitatively determined the differential particle size uptake. These results demonstrate the potential of nanoparticle-based fluorescence lifetime multiplexing in cellular studies and cell-based assays.
Low-frequency fluctuations of an atomic force microscope cantilever is capable of monitoring nanometer-scale activity of bacteria in real time Evaluation of: Longo G, Alonso-Sarduy L, Marques Rio L et al. Rapid detection of bacterial resistance to antibiotics using AFM cantilevers as nanomechanical sensors. Nat. Nanotechnol. 8(7), 522–526 (2013). The widespread use of drugs has increased the number of multidrug-resistant bacteria. Therefore, in the overall approach to managing infection, there is a critical need to develop tools that can rapidly characterize bacterial response to antibiotics. Although techniques for the
detection and characterization of bacterial resistance to antibiotics are available, the process is time consuming. In this study, Longo and coworkers demonstrated that the fluctuations of highly sensitive atomic force microscope cantilevers can be used to quickly detect quantitatively low concentrations of bacteria according to their metabolism and response to antibiotic treatment. The measurement is achieved by immobilizing live bacteria on a canti lever sensor. The movements of the bacteria cause an increase in the amplitude of the fluctuations of the sensor, which can be monitored using an atomic force microscope. The authors tested this technology
on Escherichia coli and Staphylococcus aureus and confirmed its reliability for reporting bacterial metabolism. Before the bacteria were introduced, the fluctuations were very slight; however, the fluctuations increased and appeared to become strongly dependent on bacterial activity after their introduction. Following treatment with ampicillin, the fluctuations diminished to very low values and remained as such after the antibiotic was flushed out. This suggests that the ampicillin eliminated the bacteria. In general, the application of this novel technique is not limited to bacteria, as it can be used for studying cells or protein conformational changes.
Lipid nanoparticles as ideal delivery modules for siRNA Evaluation of: Gilleron J, Querbes W, Zeigerer A et al. Image-based analysis of lipid nanoparticle‑mediated siRNA delivery, intracellular trafficking and endosomal escape. Nat. Biotechnol. 31(7), 638–646 (2013). 1910
Synthetic siRNAs have emerged as a promising therapeutic modality to silence disease-associated genes. However, while the delivery of an active form of siRNA is a complex process, it is critical in the successful development of siRNA-based drugs currently being tested in several clinical trials. Liposomes and polycationic Nanomedicine (2013) 8(12)
polymers have been used for siRNA delivery, although those techniques suffer from various levels of efficiency and toxicity, thus making them clinically unsatisfactory. By contrast, lipid nanoparticles (LNPs) are the most advanced delivery systems for siRNAs. The construct is comprised of multiple lipids that self-assemble future science group
Research Highlights News – into 60–80‑nm particles, thereby encapsulating the siRNA molec ules. Nevertheless, the underlying mechanism of LNP-mediated siRNA delivery is not yet fully understood. In this study, Gilleron et al. reported that LNPs enter cells via both constitutive and inducible pathways in a cell type-specific manner using clathrin-mediated endoc ytosis (CME) as well as macropinoc ytosis. Inhibition of CME using dynasore (a small-molecule inhibitor of dynamin), a GTPase required for membrane fission in CME or macropinocytosis using ethylisopropylamiloride reduced uptake by 50–70%,
future science group
which suggests that both pathways are required. Furthermore, the authors used time-lapse confocal fluorescence micro scopy to examine the release of siRNA into the cytosol. The results demonstrated that siRNAs delivered by LNPs are not massively released owing to the bursting of individual endosomes or the permeabilization of the limiting membrane of endosomal compartments. It is likely that such release is restricted to a limited number of siRNAs and is, therefore, undetectable using standard fluorescence microscopy methods. However, by using electron microscopy to study the release
www.futuremedicine.com
News & Views
using LNP–siRNA–gold, the authors discovered that siRNA–gold escaped from endosomes in hepatocytes in vivo and reached a level of 186.05 siRNA–gold per cell after 6 h. In general, the results reported in this work add insight to LNPmediated siRNA delivery, which may help guide the development of the next generation of delivery systems for RNAi therapeutics.
1911
