Accessible Published by De Gruyter November 9, 2012

Nanoparticle-functionalized microcapsules for in vitro delivery and sensing

Susana Carregal-Romero, Markus Ochs and Wolfgang J. Parak
From the journal Nanophotonics

Abstract

Inorganic nanoparticles such as magnetic nanoparticles, fluorescent quantum dots, and plasmonic nanoparticles can be used as building blocks for designing multifunctional systems based on polymeric capsules. The properties of the inorganic nanoparticles hereby are harnessed to provide additional functionality to the polymer capsules. Biological applications towards in vitro sensing and delivery are discussed. Examples will be given in which magnetic nanoparticles are used to direct capsules with magnetic field gradients, colloidal quantum dots are used to identify capsules via the formation of optical barcodes, and gold nanoparticles are used as light-controlled heat-sources for opening capsules and releasing macromolecules from their cavity upon optical excitation. This demonstrates that combination of inorganic nanoparticles and organic/polymeric molecules as carrier matrices allow for tailoring multifunctional hybrid particles for practical applications.

1. Introduction

Progress in biology is often influenced by the development of new assays or tools. They even can allow for monitoring cellular processes which have not been experimentally accessible before, be it due to previous limits in sensitivity, long-term stability, biocompatibility, or experimental complexity. Particle-based systems are helpful tools in this direction and have been used as contrast agents for imaging, as sensors for the detection of analytes, or as delivery vehicles in vitro and in vivo [1–4]. Inorganic nanoparticles (NPs) for example can contribute different properties based on their material composition. Fluorescent quantum dots (QDs) such as CdSe/ZnS or InP NPs can be used as tags for cellular imaging. Magnetic NPs such as Fe2O3 or Fe3O4 NPs can be used as contrast agents for magnetic resonance imaging (MRI) or can be guided in magnetic field gradients. Plasmonic NPs such as Au or Ag NPs can be used for optical sensing or for converting light into heat. To produce heat efficiently the nanoparticles have to be irradiated with light in the same wavelength range of the plasmon band absorption. The plasmon band of such NPs can be tuned easily by changing size or shape. Plasmonic NPs absorbing in the near-infrared region of the electromagnetic spectrum of the light are more suitable for biological applications since the absorption of light by tissue is minimal. Therefore, Au nanoshells, small Au aggregates or Au nanorods are convenient platforms to be used as nanoheaters [5–7]. By integrating different inorganic NPs into bigger carrier systems their properties can be combined, which thus allows for creating multifunctional objects. Polymeric polyelectrolyte capsules are one example of such a carrier system [8], which is on first order held together by electrostatic attraction and thus easily allows for integrating charged NPs of different materials [9–11]. In this review we will show three examples on how incorporation of magnetic, fluorescent, and plasmonic NPs into capsules provide them particular properties useful for in vitro delivery and sensing.

2. Polyelectrolyte capsules as universal carrier systems

Polyelectrolyte multilayer (PEM) capsules are fabricated following a bottom-up approach via Layer-by-Layer (LbL) self-assembly [12] of differently charged polyelectrolytes on top of a template particle [13, 14]. Hereby the onion-shaped LbL geometry is held together predominantly by electrostatic force. Subsequent dissolution of the template particle leads to PEM capsules, cf.Figure 1. PEM capsules have several distinct features: (i) They can carry a cargo in their cavity and other functionalities can be integrated in their PEM walls. Cargo can comprise macromolecules [16], hydrophobic drugs [17], micelles [18], or NPs [19]. In addition, walls can be modified with biological ligands or NPs. As pointed out before NPs can be fluorescent, magnetic, light mediated heaters, etc. Loading the cavity and the walls independently with several of the aforementioned entities allows for multifunctionality. (ii) The cargo inside the capsule cavity is protected within the polyelectrolyte walls and does not participate in the control over pharmacokinetics and biodistribution. Cells which have incorporated capsules are also protected from direct contact with the containing cargo. (iii) Size and charge of the PEM capsules can be easily tuned [14, 20]. Size and charge are important parameters which affect interaction with cells. Neutral or slightly negative charge helps to reduce non-specific uptake by living cells in vivo and positive charged systems favored non-specific uptake [21]. (iv) The PEM wall can be biodegradable or non-degradable and its porosity can be tuned by the number of polyelectrolyte layers and by the PEM materials. Due to this tunable porosity small molecules can diffuse in and out the cavity to bulk solution, but bigger molecules as cargo are trapped inside the cavity. The porosity of the capsules depends strongly on the number of polyelectrolyte layers and on the presence of other entities such as nanoparticles. Dong et al. studied, for example, the diffusion of different fluorophores with different hydrodynamic sizes (from 0.8 to 9 nm) for capsules made with a different number or polyelectrolyte layers [22] but changes in the LbL process such ionic strength, the polyelectrolyte composition and the addition of NPs or the pH of the environment are known to change the diffusion of analytes through the polymeric shell [22, 23]. Nevertheless, the PEM capsule shell is in general permeable for small ions such as H+ or Na+.

Figure 1 (A) Scheme of a multifunctional PEM capsule (not drawn to scale). (B) Transmission electron microscope (TEM) image of a PEM capsule with incorporated NPs in their wall (5 double layers of polystyrene sulfonate Mw≈70 kDa)/poly (allylamine hydrochloride) (Mw≈56 kDa); Au NPs with core diameter of 20 nm). The scale bar corresponds to 1 μm. Image taken from del Mercato et al. [15].

Figure 1

(A) Scheme of a multifunctional PEM capsule (not drawn to scale). (B) Transmission electron microscope (TEM) image of a PEM capsule with incorporated NPs in their wall (5 double layers of polystyrene sulfonate Mw≈70 kDa)/poly (allylamine hydrochloride) (Mw≈56 kDa); Au NPs with core diameter of 20 nm). The scale bar corresponds to 1 μm. Image taken from del Mercato et al. [15].

In Figure 1 the idealized scheme of a PEM capsule is shown. It is important to point out the different size of the NPs (hydrodynamic diameters around 10 nm) and the PEM capsules (diameters around 3–5 μm) which will be discussed in the following. The PEM wall of the capsules is remarkably thin, wherein each layer contributes between 1 and 10 nm to the thickness depending on the PE nature and on the ionic strength during the LbL process [22]. Thus typically the size of NPs incorporated in the capsule wall is bigger than the actual thickness of the plain wall. Even at diameters of a few microns PEM capsules are non-specifically incorporated by most cell lines [24, 25]. Though the actual mechanism for internalization is still not fully unraveled, most studies agree that the internalized capsules are finally located in the lysosome. For most in vitro applications there is no acute cytotoxicity [26, 27]. Even in vivo administration generates only a moderate immune reaction upon subcutaneous and mucosal administration similar to some of natural and synthetic polymer-based particles such as polylactide-co-glycolide (PLGA) [26, 28]. PEM capsules are generally stable in cell medium but there is anyway absorption of proteins due to their charged surface. Nevertheless, it can be minimized by functionalizing the surface with poly(l-lysine)-g-poly(ethylene glycol) [29]. Thus, in the case of in vitro applications capsules have sufficient biocompatibility for performing experiments over the time range of weeks. In the present review now three applications of multifunctional capsules with inorganic nanoparticles will be introduced and discussed. These examples will demonstrate how (i) magnetic NPs, (ii) fluorescent NPs, and (iii) plasmonic NPs can be harnessed to add functionality to the PEM capsules and facilitate applications for in vitro delivery and sensing.

3. Magnetic NPs for targeted local uptake and release

The idea of exploiting magnetic guidance, which uses an implanted permanent magnet or an externally applied field, to increase the accumulation of drugs at diseased sites dates back to the late 1970s. Objects possessing a magnetic moment experience a force in magnetic field gradients. In this way it is possible to direct and accumulate those objects at a designated target site. This concept has been successfully used for example for in vivo targeting of drug-loaded magnetic NPs to tumor tissue [30, 31]. As pointed out NPs can be easily incorporated in the wall of PEM capsules. In this way the existing concept of magnetic targeting could be easily transferred to capsules. As many magnetic NPs can be loaded to each capsule the resulting magnetic moment is rather high. Thus even gradients generated by magnets from a toy store are sufficient to trap capsules at desired positions of cell cultures in a model flow channel system [32]. The magnetic field gradient itself does not stimulate internalization of the capsules, but it accumulates capsules by locally trapping them. As the uptake of capsules by cells depends on their local concentration consequently at the target region cells have a higher number of internalized capsules. This can be used for active delivery of cargo to the target region. One very interesting type of “cargo” is small interference RNA (siRNA). RNA interference (RNAi) has gained increasing attention due to its remarkable potential to regulate gene expression of virtually any identifiable molecular target. In particular, gene silencing can be induced by siRNA [33]. However, this molecule can be degraded in vivo by serum or tissue nucleases and due to its small size it suffers as well from a rapid renal clearance [34]. Encapsulation might help to circumvent some of these drawbacks. The concept of magnetic targeting is very universal, and magnetic NPs can be introduced into a large variety of carrier systems, as demonstrated above for PEM capsules. Lipospheres (stabilized with a mixture of cationic lipids) are conceptually similar to capsules and can also be modified with magnetic NPs in their walls and can carry a cargo such as siRNA in their cavity. For example magnetic targeting of lipospheres was demonstrated with HeLa cells which were expressing green fluorescent protein (GFP). Lipospheres with magnetic NPs and siRNA against GFP expression were added in a flow channel system above HeLa eGFP cells in which a little magnet was placed at the target region [35], cf.Figure 2. As mentioned above, the magnetic field gradient accumulated lipospheres close to the magnet, and due to their enhanced concentration more lipospheres entered cells and thus delivered siRNA. The siRNA interacted with a target mRNA leading to suppression of GFP expression in the cells close to the magnet. SiRNA delivery could be observed by quenching of GFP fluorescence in the cells nearby the magnetic field, cf.Figure 2. Similar delivery of active compounds should be possible also with PEM capsules. Recently, the release of a fluorophore from PEM capsules loaded with magnetite upon the action of an alternating magnetic field has been achieved in water [36]. Thus, in vitro applications of magnetically triggered release from PEM capsules are expected in the future. At any rate this example demonstrates that addition of magnetic NPs to carrier systems provides them with new properties, in this case with a magnetic moment, which can be used for magnetic targeting and delivery.

Figure 2 (A) Sketch of a liposphere containing magnetic NPs in their walls and siRNA against GFP expression as cargo. Lipospheres are added to a flow channel above a cell culture of GFP expressing HeLa cells. A magnet is placed in the middle of the flow channel to define the target region for magnetically targeted delivery. (B) Optical microscopy images taken 72 h after addition of lipospheres (overlay of phase contrast and green fluorescence channel). Expression of GFP in cells close to the magnet is quenched due to the delivery of siRNA, whereas cells further away from the magnet are unaffected. Image taken from del Pino et al. [35].

Figure 2

(A) Sketch of a liposphere containing magnetic NPs in their walls and siRNA against GFP expression as cargo. Lipospheres are added to a flow channel above a cell culture of GFP expressing HeLa cells. A magnet is placed in the middle of the flow channel to define the target region for magnetically targeted delivery. (B) Optical microscopy images taken 72 h after addition of lipospheres (overlay of phase contrast and green fluorescence channel). Expression of GFP in cells close to the magnet is quenched due to the delivery of siRNA, whereas cells further away from the magnet are unaffected. Image taken from del Pino et al. [35].

Magnetic NPs embedded into PEM capsules could also act as contrast agents for MRI imaging due to their magnetic properties that can be tuned by changing the packing of the NPs within the polymeric shell. They could be useful for in vivo imaging or as theranostic agents (therapy and diagnosis) [37]. The use of PEM capsules for in vivo applications is still a matter of discussion but there is a general agreement about the importance of the wall composition to avoid toxicity effects. The use of polypeptides homopolymers or polyssacharides as polyelectrolytes and non toxic nanoparticles such as magnetite NPs will, in principal, decrease the potential toxic effects. Moreover, the size of the capsules could limit their applications. Drug delivery and vaccination applications of PEM capsules have been recently discussed by De Geest et al. [8, 38].

4. Fluorescent NPs for barcoding of capsules enabling spatially resolved sensing

Sensing of ions is important for a large variation of cell biological applications. One common detection technique is fluorescence detection of analyte-sensitive fluorophores. Such analyte-sensitive fluorophores are (often organic) fluorescence dyes, of which (in general) the fluorescence emission intensity selectively depends on the presence of a specific type of ion, such as H+, K+, Na+, Ca2+, Cl-, etc. Presence of ions can either enhance or quench the fluorescence, depending on the chemical nature of the fluorophore. There are many fluorophores available to determine the concentration of different ions such as H+ [39], K+ [40], Na+ [41], and Cl- ions [42], etc. The response of different fluorophores can (upon simultaneous excitation) only be distinguished if they emit at sufficiently different wavelengths. Although a few fluorophores can be independently detected, the number of fluorophores that can be spectrally distinguished is clearly limited by their spectral width of emission, which ultimately results in emission crosstalk and thus hinders multiplexing (Figure 3).

Figure 3 (A) Two fluorophores can be spectrally distinguished in case their wavelengths λ of emission are sufficiently different. (B) In case two fluorophores emit at similar wavelength they can’t be spectrally resolved. (C) In case the fluorophores are located at different positions x they can be resolved, even in case their spectra overlap. Fluorophores can be spatially separated by placing them in containers such as PEM capsules. In order to distinguish between different capsules they can be tagged with a fluorescent barcode on their surface. Data adopted from Abbasi et al. [43].

Figure 3

(A) Two fluorophores can be spectrally distinguished in case their wavelengths λ of emission are sufficiently different. (B) In case two fluorophores emit at similar wavelength they can’t be spectrally resolved. (C) In case the fluorophores are located at different positions x they can be resolved, even in case their spectra overlap. Fluorophores can be spatially separated by placing them in containers such as PEM capsules. In order to distinguish between different capsules they can be tagged with a fluorescent barcode on their surface. Data adopted from Abbasi et al. [43].

One suggested possibility of circumventing this problem is based on spatial discrimination instead of spectral resolution [43]. The concept of spatial discrimination of different ion-sensitive fluorophores is straightforward. In case each different ion-sensitive fluorophore can be provided with a unique tag (which might be a fluorophore or inorganic NPs), and in case the average distance between different fluorophores is higher than the optical resolution limit, individual fluorophores can be separately addressed and read-out. PEM capsules are promising systems in this direction due to the porosity of their wall and the possibility of loading different parts of their geometry with fluorophores. The inner cavity can be loaded with the ion-sensitive fluorophores, and the wall of the capsules with a fluorescent barcode. Micrometer sized PEM capsules can be clearly individually resolved, and thus fluorescence of the ion-sensitive fluorophores of each capsule can be individually recorded. The fluorescent barcode within the PEM walls allows for differentiation of the individual capsules and subsequent identification of the different sensor fluorophores.

In order to sense ions they must be able to traverse the capsule walls and reach the ion-sensitive fluorophores in the capsule cavity. As the wall of PEM capsules is porous [44] this is generally no problem. Porosity depends for example on the used polyelectrolyte materials and the number of polyelectrolyte layers [22, 45]. A bigger problem is keeping the ion-sensitive fluorophores inside the capsules. In order to prevent their diffusion through the pores of the PEM walls they can be linked to macromolecules such as dextran. In addition, the number of deposited layers influences the permeability of the PEM walls. Besides the analyte sensitive fluorophore, which emits e.g., in the green an additional reference fluorophore, whose fluorescence does not depend on the ion concentration and which emits in a different spectral field e.g., in the red can be introduced into the same capsule. This allows for radiometric measurements, i.e., ion concentrations are not measured in terms of absolute fluorescence intensities but via analyzing emission intensity ratios of the ion sensitive fluorophores to the reference fluorophores [16]. Taking advantage of their fluorescence stability against photobleaching and their sharp emission band, mixtures of different quantum dots (QDs) can be used as barcode for every type of capsule. QDs in general have been proven as versatile barcodes allowing for many different combinations [46–48]. However, the principle of spatial discrimination between different types of capsules only works if the fluorescent barcodes (for distinguishing the capsules) do not interfere with the fluorescence for the ion-sensitive fluorophores inside the capsule cavity (for determining the ion-concentration). As mentioned above, fluorophores inside the capsule cavity tend to diffuse through the pores of the PEM walls. Though this can be reduced by attachment to macromolecules the fluorophore distribution inside the capsules is not homogeneous and fluorophores tend to stick to the inner wall [16]. Thus they would interfere with the barcode. In order to circumvent this problem double wall capsules [49] can be used, in which the ion-sensitive fluorophores are retained in the inner cavity, and the barcode is situated in the outer PEM wall, cf.Figure 4 [50]. In Figure 5 a mixture of three different types of such PEM capsules, loaded with ion-sensitive fluorophores against H+, Na+, and K+ with orange, green, and yellow barcode, respectively, are shown. Due to the barcode the different types of capsules can be clearly distinguished. This also applies for the fluorescence read-out of the distinct fluorophores depending on their respective analyte concentration [43]. Thus the principle of multiplexed ion detection could be demonstrated.

Figure 4 Double wall PEM capsule with a first inner capsule which is filled with a green fluorophore linked to dextran, and an outer wall which is labeled with red fluorescent QDs. (A) green fluorescence, (B) transmission, and (C) red fluorescence channel. (D) overlay of all channels. Scale bars correspond to 10 μm.

Figure 4

Double wall PEM capsule with a first inner capsule which is filled with a green fluorophore linked to dextran, and an outer wall which is labeled with red fluorescent QDs. (A) green fluorescence, (B) transmission, and (C) red fluorescence channel. (D) overlay of all channels. Scale bars correspond to 10 μm.

Figure 5 Multiplexed measurements of ions with barcoded PEM capsules. (A) Three different types of capsules have been synthesized. Capsules were co-loaded in their cavities with the dextran-modified ion sensitive fluorophores FITC, SBFI, and PBFI, and with dextran-modified reference fluorophore Dy647 [50]. Thus fluorescence originating from the cavity is sensitive to pH, Na+, and K+, respectively. The capsules were labeled with a quantum dot based fluorescent barcode (orange, green, and yellow) on their outermost surface, (B) Fluorescence image of a mixture of the three different types of capsules. Due to the barcodes all types of capsules can be read-out independently, which allows for multiplexed ion detection. The scale bar corresponds to 5 μm. Figure adopted from Abbasi et al. [43].

Figure 5

Multiplexed measurements of ions with barcoded PEM capsules. (A) Three different types of capsules have been synthesized. Capsules were co-loaded in their cavities with the dextran-modified ion sensitive fluorophores FITC, SBFI, and PBFI, and with dextran-modified reference fluorophore Dy647 [50]. Thus fluorescence originating from the cavity is sensitive to pH, Na+, and K+, respectively. The capsules were labeled with a quantum dot based fluorescent barcode (orange, green, and yellow) on their outermost surface, (B) Fluorescence image of a mixture of the three different types of capsules. Due to the barcodes all types of capsules can be read-out independently, which allows for multiplexed ion detection. The scale bar corresponds to 5 μm. Figure adopted from Abbasi et al. [43].

Non-specific response of several ion-sensitive fluorophores (e.g., the fluorophores SBFI and PBFI for the detection of Na+ and K+ interfere with pH) imposes a technical complication for determining specific ion concentrations [43]. However, multiplexed detection as demonstrated above can help to circumvent this problem. Let us assume a situation in which concentrations of 3 ions in solution is to be detected using 3 different ion-sensitive fluorophores. Though each fluorophore predominantly will respond only to one type of ion, it still also will slightly respond to changes in concentration of the other ion species. For example fluorophores specific to Na+ typically also respond slightly to K+, and vice versa. However, as the ion-sensitive fluorophores are confined in different capsules all three types of fluorophores can be read-out in parallel. Thus there are 3 unknowns (ion concentrations), but also 3 read-outs (due to the multiplexed detection), which allows for determining all 3 unknowns via a calibration curve [43].

In order to demonstrate potential applications of capsule-based ion-sensing in vitro in the following an example based on pH-sensitive capsules, which are loaded with the pH-sensitive fluorophore SNARF in their cavity, is given. SNARF fluoresces in the green-yellow and red at acidic and alkaline pH, respectively. Thus capsules in the slightly alkaline extracellular medium show red fluorescence, whereas capsules which have been incorporated by cells and are located in the acidic lysosome are fluorescent in yellow [25], as can be seen in Figure 6. Internalized capsules reside in the lysosome over time. This automatically involves the fact that intracellular sensing as shown here is actually sensing of the environment of the lysosome, and not of the cytosol. At any rate, addition of certain pharmaceutical agents, such as Monensin, Chloroquine, and Bafilomycin changes the lysosomal pH. By time-resolved recording the fluorescence of the internalized capsules changes in pH upon stimulating cells with pharmaceutical agents can be observed. In particular this allows for recording of kinetics, i.e., to determine how fast the pH in the lysosome changes upon addition and removal of pharmaceutical agents [51].

Figure 6 (A) Cells have been incubated with pH-sensitive PEM capsules, which have green-yellow/red fluorescence in acidic/alkaline environment. Some of the capsules are spontaneously incorporated by the cells and are transported to the acidic lysosome and thus are fluorescent in the green-yellow, whereas capsules remaining in the slightly alkaline cell medium fluoresce in red. pH changes in the lysosome upon stimulation of cells with pharmaceutical agents can be traced by monitoring the color of fluorescence of the internalized capsules. (B) Overlay of microscopy images (phase contrast, yellow fluorescence, red fluorescence) before and after addition of an agent (in this case Chloroquine), which increases the pH inside the lysosome. (C) The ratio of red to yellow fluorescence (Ir/Iy) of the capsules depends on the surrounding pH. Reference capsules in the slightly alkaline extracellular medium act as control to compensate for photobleaching. Upon stimulation with a pharmaceutical agent the pH inside the lysosome may change, as can be seen by changes in the Ir/Iy-ratio of internalized capsules. Image adopted from Rivera Gil et al. [51].

Figure 6

(A) Cells have been incubated with pH-sensitive PEM capsules, which have green-yellow/red fluorescence in acidic/alkaline environment. Some of the capsules are spontaneously incorporated by the cells and are transported to the acidic lysosome and thus are fluorescent in the green-yellow, whereas capsules remaining in the slightly alkaline cell medium fluoresce in red. pH changes in the lysosome upon stimulation of cells with pharmaceutical agents can be traced by monitoring the color of fluorescence of the internalized capsules. (B) Overlay of microscopy images (phase contrast, yellow fluorescence, red fluorescence) before and after addition of an agent (in this case Chloroquine), which increases the pH inside the lysosome. (C) The ratio of red to yellow fluorescence (Ir/Iy) of the capsules depends on the surrounding pH. Reference capsules in the slightly alkaline extracellular medium act as control to compensate for photobleaching. Upon stimulation with a pharmaceutical agent the pH inside the lysosome may change, as can be seen by changes in the Ir/Iy-ratio of internalized capsules. Image adopted from Rivera Gil et al. [51].

In the future similar assays may offer a convenient tool for recording changes in the ion composition inside the lysosome, in case cells are fed not only with one type of capsules (as the pH-sensitive ones as shown above), but with several barcoded capsules which are sensitive for different types of ions. Clearly one limitation of this technique is the fact that capsules inside cells are confined to the lysosome and not freely mobile in the cytosol. Pharmaceutical research offers several approaches for transferring molecules from the lysosome to the cytosol, such as the proton sponge effect of polyethyleneimine (PEI) [52], which also might be used for translocation of capsules. We on the other hand want to point out that also in this case inorganic NPs might offer and interesting solution, as will be explained in the next paragraph.

5. Plasmonic nanoparticles – light-triggered release

Macromolecules or particles (such as PEM capsules) internalized via the endocytic pathway are subsequently routed to lysosomes for enzymatic degradation [53]. Thus, disruption or timely permeabilization of the endosomal membrane is a prerequisite for their cytosolic translocation. Strategies in this direction involve cell penetrating peptides [54], pH-sensitive carriers [55], or the proton sponge effect of PEI [56]. Recently, several groups have also proposed release by local heating. Plasmonic NPs, in particular Au NPs, can be optically excited to a resonance in which a collective motion of free electrons occurs, the so-called surface plasmon [57]. Energy is transferred ultrafast from the electrons to the crystal lattice in the form of phonons and the occurring heat dissipates to the local environment. In other words, plasmonic NPs can efficiently convert light into local heat. This effect has been for example used to locally destroy tissue [58, 59]. Other groups have used this strategy for opening containers, such as PEM capsules [10, 60]. For this purpose Au NPs are integrated in the PEM wall of the capsules. Illumination at the surface plasmon resonance frequency causes heating of the NPs, which in turn locally disintegrates the PEM wall and also perforates the membrane of the surrounding lysosome in which the capsules are located [61–63], resulting in release of the molecules from the capsule cavity to the cytosol. Release to the cytosol is indicated by the fact that the released cargo is homogeneously distributed over the whole body of the cells (excluding the nucleus), and that released pH indicators (cf. pH detection with SNARF in the paragraph above) demonstrate transfer from an acidic compartment (lysosome) to a neutral compartment (cytosol) [64]. Before discussing obvious limitations of this technique we first point out its potentials. Light-mediated release of macromolecules from capsules can be seen as an extension of the concept of caged-calcium [65, 66], where Ca2+ ions are released from chelators upon a light trigger. Caged compounds have been proven to be a very valuable tool for in vitro investigations, where onset of a biological action can be externally triggered by light-mediated release of a specific compound. Typically caged compounds are rather available from small molecules. PEM capsules modified with plasmonic NPs in their walls and macromolecular cargo in their cavity can extend this concept for the light-triggered release (in vitro) of macromolecules. Opening of capsules works on the basis of individual capsules. In case both the capsules and the light-pointer have micrometer size, the capsules can be opened one by one (with complete control) and the whole process of irradiation and release can be observed with optical microscopy. If cells are loaded with capsules bearing different macromolecules in their cavities subsequent opening causes controlled mixing of the released macromolecules in the cytosol, cf.Figure 7 [64].

Figure 7 (A) Cells were incubated with a mix of Au NP modified capsules which were loaded either with blue or with a red fluorescence labeled dextran. The shown microscopy images are the red and blue fluorescence channel and an overlay of both with the transmission channel. (B) First the blue capsules inside cells were opened with the light pointer and subsequent release of blue fluorescent dextran to the cytosol can be observed. Red fluorescent dextran is still confined to the capsules. (C) In a second step also the red capsules were opened with the light pointer and thus red fluorescent dextran was released to the cytosol, where it mixed with the blue fluorescent dextran. The scale bars represent 10 μm. Arrows pointed at the irradiated and thus opened capsules. Image adapted from Carregal-Romero et al. [64].

Figure 7

(A) Cells were incubated with a mix of Au NP modified capsules which were loaded either with blue or with a red fluorescence labeled dextran. The shown microscopy images are the red and blue fluorescence channel and an overlay of both with the transmission channel. (B) First the blue capsules inside cells were opened with the light pointer and subsequent release of blue fluorescent dextran to the cytosol can be observed. Red fluorescent dextran is still confined to the capsules. (C) In a second step also the red capsules were opened with the light pointer and thus red fluorescent dextran was released to the cytosol, where it mixed with the blue fluorescent dextran. The scale bars represent 10 μm. Arrows pointed at the irradiated and thus opened capsules. Image adapted from Carregal-Romero et al. [64].

Obviously there are also clear limitations for this technology. First, the power of the light-pointer has to be controlled very well. Simply speaking heating is trivial, but controlled heating is more complicated. In case too much power is applied water is evaporated and the resulting gas bubbles destroy cells. Fortunately the complex cellular environment reduces bubble formation [6], but nevertheless overheating remains the main risk. The mode of laser-tissue interactions depends on how the light energy is applied. At low laser energy flow and long exposure, exposure can lead to photochemical or photothermal interactions. Confining the laser into short pulses can cause intense heating followed by water phase change in biological systems, i.e., bubble formation and photoablation and further increase of the laser energy or shorter pulses eventually may lead to plasma-induced ablation and photodisruption [67]. In this context, for capsules modified with plasmonic NPs the simultaneous opening of many capsules via homogeneous illumination is technically challenging due to the shifts of the laser power. Moreover, the cargo molecules in the cavity of the capsule could be damaged upon light-mediated heating. Bioactive molecules should keep their functionally upon laser irradiation. However, localized heating could lead to the destruction of the active part of the molecules to be released. In order to study the possible laser damage of the capsule payload, green fluorescent protein (GFP) has been released into the cytosol [64]. As fluorescence of released GFP could be observed one can conclude that at least part of the released GFP and its function remained intact. The local heating produced after light absorption of the gold NPs in the capsule walls can be thus tuned in a way that it is enough to disrupt the PEM walls of the capsules (and the surrounding lysosomal membrane), but does not damage the whole amount of released protein. Clearly quantitative release as the current state-of-the-art is not possible, due to inhomogeneous loading of the capsules with cargo in their cavity, variations in Au NP concentrations in the PEM walls, and possible partial destruction of the cargo molecules by heat. Thus PEM capsules are best suited for the release of molecules which can make an all-or-nothing response, which does not quantitatively depend on the exact amount of released functional molecules. Kinetics of drug release by light responsive capsules is as well difficult to perform since the increase of fluorescence in the cytosol when cargo molecules are released (if they were not quenched or needed the action of enzymes) is immediate. Only molecules that develop fluorescence with the time once they are located into the cytosol are suitable to perform kinetic studies due to the limitation of the optical microscopes themselves.

6. Outlook

In these perspectives we tried to point out how inorganic NPs can be useful building blocks for modifying organic/polymeric carrier matrices with additional functionalities. This helps for creating new multifunctional hybrid materials, which have a clear potential as tools for in vitro sensing and delivery. On purpose this perspective is limited on in vitro applications, as any in vivo applications would involve problematic points such as cytotoxicity issues, biodistribution, etc. to a much higher extent. On the other hand, three examples have been given on how these hybrid capsules could serve as interesting tools for cell culture experiments.

This work has been supported by BMBF Germany (ERANET grant Nanosyn to WJP). SCR is grateful to the Junta Andalucía for a fellowship.

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Received: 2012-3-21
Accepted: 2012-5-22
Published Online: 2012-11-09
Published in Print: 2012-11-01

©2012 by Science Wise Publishing & De Gruyter Berlin Boston