Evaluating Cellular Drug Uptake with Fluorescent Sensor Proteins

Scarabelli, Silvia; Tan, Kui‐Thong; Griss, Rudolf; Hovius, Ruud; D'Alessandro, Pier Luca; Vorherr, Thomas; Johnsson, Kai

doi:10.1021/acssensors.7b00331

Cited by 23 publications

(21 citation statements)

References 40 publications

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“…Therefore, complex approaches such as radio-labeled molecules or in vitro models with synthetic membranes must be used to accomplish the quantification. 15 Improving the cell uptake of small molecules Prodrug strategy: Lipophilicity is one the main parameters that determine cell uptake by simple diffusion. The lipophilic character of a molecule can be enhanced by means of chemical modifications; the simplest way involves masking polar groups such as carboxylic acids, phosphates, and other charged groups, by forming esters.…”

Section: Cellular Uptake Of Small Moleculesmentioning

confidence: 99%

Cellular Uptake of Nanoparticles versus Small Molecules: A Matter of Size

2018

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The primary function of the cell membrane is to protect cells from their surroundings. This entails a strict regulation on controlling the exchange of matter between the cell and its environment. A key factor when considering potential biological applications of a particular chemical structure has to do with its ability to internalize into cells. Molecules that can readily cross cell membranes are frequently needed in biological research and medicine, since most therapeutic entities are designed to modulate intracellular components. However, the design of molecules that do not penetrate cells is also relevant toward, for example, extracellular contrast agents, which are most widely used in clinical diagnosis. Small molecules have occupied the forefront of biomedical research until recently, but the past few decades have seen an increasing use of larger chemical structures, such as proteins or nanoparticles, leading to unprecedented and often unexpectedly novel research. Great achievements have been made toward understanding the rules that govern cellular uptake, which show that cell internalization of molecules is largely affected by their size. For example, macromolecules such as proteins and nucleic acids are usually unable to internalize cells. Intriguingly, in the case of nanoparticles, larger sizes seem to facilitate internalization via endocytic pathways, through which the particles remain trapped in lysosomes and endosomes. In this Account, we aimed at presenting our personal view of how different chemical structures behave in terms of cell internalization due to their size, ranging from small drugs to large nanoparticles. We first introduce the properties of cell membranes and the main mechanisms involved in cellular uptake. We then discuss the cellular internalization of molecules, distinguishing between those with molecular weights below 1 kDa and biological macromolecules such as proteins and nucleic acids. In the last section, we review the biological behavior of nanoparticles, with a special emphasis on plasmonic nanoparticles, which feature a high potential in the biomedical field. For each group of chemical structures, we discuss the parameters affecting their cellular internalization but also strategies that can be applied to achieve the desired intracellular delivery. Particular attention is paid to approaches that allow conditional regulation of the cell internalization process using external triggers, such as activable cell penetrating peptides, due to the impact that these systems may have in drug delivery and sensing applications. The Account ends with a "Conclusions and Outlook" section, where general lessons and future directions toward further advancements are briefly presented.

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Section: Cellular Uptake Of Small Moleculesmentioning

confidence: 99%

Cellular Uptake of Nanoparticles versus Small Molecules: A Matter of Size

2018

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“…Another approach to measure cytosolic concentrations, based on an intracellular FRET system, has been demonstrated to work for detection of p53/HDM2 inhibitors. [9] This sensor, exquisitely designed by GGS-spacers and poly-Pro residues to optimally arrange for a FRET system, contains the target and a moderately active binding peptide to enable interaction in an intramolecular fashion (Fig. 2).…”

Section: Cellular Uptake Of Peptidesmentioning

confidence: 99%

“…The fusion protein consists of a p53 peptide on YPet connected through a flexible Pro-linker to CFP plus the target HDM2. [9] The free analyte in solution (green ellipsoid) displaces the p53-peptide intramolecular ligand and shifts the sensor to the open conformation as a result reducing the FRET efficiency.…”

Section: Oral Bioavailability Of Peptidesmentioning

confidence: 99%

Important Considerations Related to Permeability of Peptides

Vorherr

2021

Chimia

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This review on intracellular delivery and oral bioavailability of peptides reflects a number of principal investigations at Novartis. Our studies were aimed at either understanding features enabling peptides to interfere with intracellular protein–protein interactions, or to achieve a more patient-friendly delivery by the oral route. In the light of these objectives, we have also spent some effort on assay development to come up with alternative methods for monitoring cellular peptide uptake. This summary of our insights is intended to help in the assessment and development of peptide therapeutics requiring membrane transition

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“…Despite their inherent simplicity, identification of potential split reporter proteins, and their appropriate dissection sites is limited. Split reporter proteins currently available include luciferase (Paulmurugan & Gambhir, ; Remy & Michnick, ), fluorescent proteins (Fan et al, ; Hu & Kerppola, ; Tchekanda, Sivanesan, & Michnick, ), beta‐lactamase (Galarneau, Primeau, Trudeau, & Michnick, ), protease (Wehr et al, ), and others (Aranko, Wlodawer, & Iwai, ; Camacho‐Soto, Castillo‐Montoya, Tye, & Ghosh, ; Chelur & Chalfie, ; Dochow, Krumm, Crowe, Moore, & Plemper, ; Engelen & Merkx, ; Freudenthal, Gakhar, Ramaswamy, & Washington, ; Griss et al, ; Guo et al, ; Mabe, Nagamune, & Kawahara, ; Massoud, Paulmurugan, & Gambhir, ; Merkx, Golynskiy, Lindenburg, & Vinkenborg, ; Muller, Kries, Csuhai, Kast, & Hilvert, ; Nirantar, Li, Siau, & Ghadessy, ; Pelletier, Campbell‐Valois, & Michnick, ; Scarabelli et al, ; Slaska‐Kiss, Timar, & Kiss, ; Tafelmeyer, Johnsson, & Johnsson, ; Yu, Griss, Schena, & Johnsson, ). Notwithstanding, none has fulfilled AAAA criteria when used in diagnostic testing, due to aforementioned reasons.…”

Section: Introductionmentioning

confidence: 99%

Split trehalase as a versatile reporter for a wide range of biological analytes

Drikic

Buck

2018

Biotech & Bioengineering

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In health care, biosensors are envisioned as universal diagnostic devices with AAAA characteristics (i.e., available for anything, anywhere, anytime, to anyone). Despite numerous attempts to develop such a diagnostic device, none have managed to fulfill all four criteria and be commercialized. Glucometers, the most successful class of biosensor currently marketed monitor blood glucose concentrations. Their performance in clinical samples, including sensitivity and specificity, has been optimized and they are small and relatively inexpensive. We aimed to develop a technology that uses this existing biosensor, but adds versatility in detection of a wide range of analytes. Herein, we report the periplasmic trehalase of E. coli as a novel split enzyme reporter capable of converting a wide variety of analytes into glucose. Conditional complementation of trehalase fragments induced by detection of analytes, resulting in trehalose hydrolysis and glucose production, was used to detect antibodies and bacterial cells. We also demonstrated retention of split TreA activity in undiluted clinical samples. In conclusion, a trehalase-based biosensor platform offers a versatile and convenient method for point-of-care applications as it does not require sample preparation or handling and can be integrated with existing glucometers or sensors.

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Evaluating Cellular Drug Uptake with Fluorescent Sensor Proteins

Cited by 23 publications

References 40 publications

Cellular Uptake of Nanoparticles versus Small Molecules: A Matter of Size

Cellular Uptake of Nanoparticles versus Small Molecules: A Matter of Size

Important Considerations Related to Permeability of Peptides

Split trehalase as a versatile reporter for a wide range of biological analytes

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