Polyelectrolyte Capsules As Carrier Systems For Local Sensing

Sensing based on optical measurements is very attractive as it allows the transduction of chemical concentration information into optical signals which can be quantified. Fluorescent indicators are a class of fluorophores whose spectral properties are sensitive to a substance (the analyte) of interest. Numerous indicators are commercially available for a variety of analytes, including Ca²⁺, Mg²⁺, Cl^-, H⁺, Na⁺, and O₂. Such analyte-sensitive fluorophores can be integrated into polymer capsules. Due to the size-dependent permeability of the walls of the polymer capsules, analyte-sensitive fluorophores of high molecular weight can be maintained in their cavity, whereas analyte molecules of low molecular weight can diffuse in and out freely. A microcapsule-based pH-sensor system using the seminaphthorhodafluor dye (SNARF-1) has already been described (fig 8a). Whereas capsules in the alkaline cell medium are fluorescent in the red, capsules which have been incorporated into cells inside acidic compartments are fluorescent in the green (fig 8a). Such pH-sensitive capsules present an interesting tool for high throughput quantification of cellular uptake. Besides the fact that naturally incorporated capsules are trapped inside intracellular compartments and thus are not in contact with the cytosol – a problem which will be discussed later – there are several advantages to embedding analyte-sensitive fluorophores into capsules. Firstly, long term measurements could be achieved, as the fluorophores inside the capsules are protected against enzymatic degradation and the cell is protected from the free fluorophore. Secondly, as many fluorophores are embedded in each capsule, there is a high local fluorophore concentration, which enhances their sensitivity (fig 8c).

Fig. 8. Capsule-based pH sensor. (a) Capsules are loaded with the pH-sensitive fluorophore SNARF in the cavity, which fluoresces green in acidic and red in alkaline environments. (b) SNARF-molecules inside capsules change their color from red in the extracellular alkaline environment of a culture medium to green upon internalization of a capsule into acidic intracellular compartments. (c) When the same number of SNARF molecules is either microinjected into a cell or introduced into a capsule carrier, the SNARF is diluted over the whole cytosol in the first case or locally concentrated inside the capsule in the second case. Local concentration of the SNARF leads to higher signal-to-noise ratios.

Most importantly, in contrast to alternative technologies such as the probes encapsulated by biologically localized embedding (PEBBLE) system, capsule-based sensors do, in principle, allow for multiplexed measurements. This is based on the fact that capsules can be functionalized with fluorescent molecules at two distinct positions, in their walls and in their cavities. In this approach, fluorophores sensitive to different analytes are loaded into the cavities of different capsules and the walls of each capsule are fluorescently labeled with a barcode (fig 9a). The color of the capsule wall would allow for identification of each capsule and thus provide the information for which analyte this particular capsule is sensitive. The local analyte concentration could be derived upon recording the fluorescence resulting from the cavity. As fluorophores for sensing different analytes are locally separated by embedding them in different capsules, spectral overlap between the different fluorophores is no longer a problem. Presumably, this concept would allow for the detection of multiple analytes in parallel in the cytosol of single cells (Fig 9b).

Fig. 9. Multiplexed sensing. (a) Capsules with semiconductor NPs as barcodes embedded in their walls and analyte-sensitive fluorophores inside their cavities. The fluorescence of the wall (here shown with blue, green, or red fluorescent NPs) is used to identify which type of analyte-sensitive fluorophore is loaded in the capsule cavity and thus to which analyte the sensor responds. (b) By loading different sensor capsules into one cell, different analytes can be detected in parallel.

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Fig. 9. Multiplexed sensing. (a) Capsules with semiconductor NPs as barcodes embedded in their walls and analyte-sensitive fluorophores inside their cavities. The fluorescence of the wall (here shown with blue, green, or red fluorescent NPs) is used to identify which type of analyte-sensitive fluorophore is loaded in the capsule cavity and thus to which analyte the sensor responds. (b) By loading different sensor capsules into one cell, different analytes can be detected in parallel.

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However, as mentioned, the delivery of capsules to the cytosol remains a fundamental problem for multiplexed measurement of different analytes inside cells. Though capsules can be directly introduced to the cytosol by electroporation, delivery to the cytosol via active capsule incorporation by cells would be preferable. However, as mentioned in earlier, capsules taken up by cells are stored inside intracellular compartments. One way of direct delivery to the cytosol might be the modification of the capsule surface with virus-derived ligand molecules, such as TAT-peptides which have been successfully used for the delivery of nanoparticles into cells. An alternative strategy is presented in fig 10. By repeated coprecipitation, double-shell capsules can be synthesized. The inner capsule could now be a sensor capsule with a fluorescent barcode in the walls and analyte-sensitive fluorophores in the cavity, while the wall of the outer capsule could be functionalized with noble metal nanoparticles. Light-illumination of such double-shell capsules inside intracellular compartments would lead to local heat generation in the metal particles. Therefore, it can be speculated that the heat would be sufficient to rupture and permeate the outer wall of the capsules as well as the membrane of the vesicular compartment in which the double-shell capsule is trapped. In this way, the intact inner sensor capsule would be released into the cytosol. Although this concept still has to be proved experimentally, there are experimental data that support the idea. Indeed, light-illumination of single metal particle-functionalized capsules not only permeates the capsule walls, but also the surrounding membrane of incorporated capsules, as demonstrated upon release of fluorescent cargo from the capsule cavity to the cytosol. Although vesicular membranes around the capsules must have been locally disintegrated, cells have been demonstrated to tolerate this treatment.

Fig. 10. Release of capsules into the cytosol. Double-shell capsules could act as a sensor capsule with a fluorescent barcode in the inner capsule wall and analyte sensitive fluorophores in the central cavity, and noble metal NPs in the outer capsule wall. Illumination of the capsules would cause heating of the metal NPs followed by disintegration of the outer wall and release of the intact inner capsule.