Evaluating Nanoparticle Sensor Design for Intracellular pH Measurements

Benjaminsen, Rikke Vicki; Sun, Honghao; Henriksen, Jonas Rosager; Christensen, Niels Jørgen; Almdal, Kristoffer; Andresen, Thomas Lars

doi:10.1021/nn201643f

Cited by 166 publications

(202 citation statements)

References 49 publications

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“…Image analysis was performed as described previously using a pixel based method [34,36]. Briefly, image processing was used in order to determine which pixels are actual signal from the nanosensor and the included pixels were then converted to pH via the calibration curve.…”

Section: Ratiometric Ph Measurements In the Lysosomal Compartmentmentioning

confidence: 99%

“…We therefore measured the lysosomal pH in cells incubated with PEI 1.8kDa, DOPE-PEI, and bPEI 25kDa using a triple-labeled ratiometric nanosensor that we have developed [34,36,37] to compare the lipid conjugated PEI with conventional PEIs. This nanosensor with the two pH-sensitive fluorophores Oregon Green and fluorescein and the pH-insensitive fluorophore rhodamine B is superior to earlier reported pH sensors with respect to the sensitivity range (pH 3.2-7.0), especially when obtaining measurements in the lysosomes [36].…”

Section: Lysosomal Phmentioning

confidence: 99%

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Elucidating the role of free polycations in gene knockdown by siRNA polyplexes

et al. 2016

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Future improvements of non-viral vectors for siRNA delivery require better understanding of intracellular processing and vector interactions with target cells. Here, we have compared the siRNA delivery properties of a lipid derivative of bPEI 1.8kDa (DOPE-PEI) with branched polyethyleneimine (bPEI) with average molecular weights of 1.8kDa (bPEI 1.8kDa) and 25 kDa (bPEI 25kDa). We find mechanistic differences between the DOPE-PEI conjugate and bPEI regarding siRNA condensation and intracellular processing. bPEI 1.8kDa and bPEI 25kDa have similar properties with respect to condensation capability, but are very different regarding siRNA decondensation, cellular internalization and induction of reporter gene knockdown. Lipid conjugation of bPEI 1.8kDa improves the siRNA delivery properties, but with markedly different 2 formulation requirements and mechanisms of action compared to conventional PEIs. Interestingly, strong knockdown using bPEI 25kDa is dependent on the presence of a free vector fraction which does not increase siRNA uptake. Finally, we have investigated the effect on lysosomal pH induced by these vectors to elucidate the differences in the proton sponge effect between lipid conjugated PEI and conventional PEI: Neither DOPE-PEI nor bPEI 25kDa affected lysosomal pH as a function of time, underlining that the possible proton sponge effect is not associated with changes in lysosomal pH.

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Section: Ratiometric Ph Measurements In the Lysosomal Compartmentmentioning

confidence: 99%

Section: Lysosomal Phmentioning

confidence: 99%

Elucidating the role of free polycations in gene knockdown by siRNA polyplexes

et al. 2016

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“…Notably, the pH nanosensor synthesized here utilized two pH-sensitive fluorophores, fluorescein and OG, with respective pKa values of 4.6 and 6.4 for improved sensitivity at lower pH ranges of interest (Figure 2A), which was required to probe lysosomal pH levels. 32 Using the ratio of the fluorescence of the pH-sensitive fluorophores to the pH-insensitive fluorophore, we calibrated a standard curve for intracellular pH readings ( Figure 2C) that could be quantified in a high-throughput manner through flow cytometry.…”

Section: Discussionmentioning

confidence: 99%

“…22,30,31 More sensitive ratiometric pH probes have recently been developed for non-gene delivery applications using fluorescent polymers and have shown the importance of using multiple pH-sensitive fluorophores to effectively probe the lysosomal pH range. 32,33 Specifically for gene delivery applications, we aimed to investigate the local pH of the exogenously delivered DNA, not the local pH of delivery polymers that may dissociate from the DNA. Here, we report a triple-fluorophore-labeled plasmid DNA-based pH nanosensor with improved sensitivity at lysosomal pH used for probing cellular pH of exogenously delivered DNA to individual cells and cell populations following polymeric gene delivery.…”

Section: Introductionmentioning

confidence: 99%

A Triple-Fluorophore-Labeled Nucleic Acid pH Nanosensor to Investigate Non-viral Gene Delivery

et al. 2017

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There is a need for new tools to better quantify intracellular delivery barriers in high-throughput and high-content ways. Here, we synthesized a triple-fluorophore-labeled nucleic acid pH nanosensor for measuring intracellular pH of exogenous DNA at specific time points in a high-throughput manner by flow cytometry following non-viral transfection. By including two pH-sensitive fluorophores and one pH-insensitive fluorophore in the nanosensor, detection of pH was possible over the full physiological range. We further assessed possible correlation between intracellular pH of delivered DNA, cellular uptake of DNA, and DNA reporter gene expression at 24 hr post-transfection for poly-L-lysine and branched polyethylenimine polyplex nanoparticles. While successful transfection was shown to clearly depend on median cellular pH of delivered DNA at the cell population level, surprisingly, on an individual cell basis, there was no significant correlation between intracellular pH and transfection efficacy. To our knowledge, this is the first reported instance of high-throughput single-cell analysis between cellular uptake of DNA, intracellular pH of delivered DNA, and gene expression of the delivered DNA. Using the nanosensor, we demonstrate that the ability of polymeric nanoparticles to avoid an acidic environment is necessary, but not sufficient, for successful transfection.

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“…This is important as has recently been reported, and the use of two fluorophores was described to achieve an intracellular pH range of 3.2-7.0. (50) Nanowire sensors can utilise the measurement of conductivity change that takes place when a macromolecule binds to the nanowire surface. Figure 13 shows how a nanowire can be used to create antigen-antibody nanosensors, in this case, for the detection of a microorganism.…”

Section: Towards Nanoparticlesmentioning

confidence: 99%

Micro- and Nanosensors for Medical and Biological Measurement

Rolfe¹

2012

Sensors and Materials

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Micro-and nanosensors have evolved rapidly in the last few decades and they have expanding roles within biology and medicine, where measurement science and technology is of key importance. The targets for measurement include a huge number of simple and complex molecules, physical quantities such as pressure, force, displacement and flow, and electrical and magnetic phenomena arising from the heart, brain, muscles and nerves. Routine clinical care of patients currently benefits from the use of macro-and microscale sensors based on electrical, electrochemical, acoustic, piezoelectric and optical principles. Disposable electrodes for recording biopotentials, such as the electrocardiogram and electroencephalogram, are common, whereas invasive electrochemical and optical fibre sensors for pressure, blood gases and pH are useful in intensive care. Microscale immobilised enzyme glucose sensors are largely confined to the analysis of small blood samples, their invasive use still facing technical challenges. Sensors constructed to the nanoscale using quantum dots and carbon nanotubes are now rapidly emerging, being aimed at more complex biomolecules such as DNA. Nanoparticles in general and surface-enhanced Raman spectroscopy also play important roles in these developments. The impact of micro-and nanosensors on the fundamental understanding of major biomedical challenges and on clinical diagnosis and care are highlighted here.

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Evaluating Nanoparticle Sensor Design for Intracellular pH Measurements

Cited by 166 publications

References 49 publications

Elucidating the role of free polycations in gene knockdown by siRNA polyplexes

Elucidating the role of free polycations in gene knockdown by siRNA polyplexes

A Triple-Fluorophore-Labeled Nucleic Acid pH Nanosensor to Investigate Non-viral Gene Delivery

Micro- and Nanosensors for Medical and Biological Measurement

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