Exploring arrays of vertical one-dimensional nanostructures for cellular investigations

High-aspect-ratio objects with nanoscale diameters and microscale heights are generally referred to as one-dimensional (1D) nanostructures (NSs). They were originally developed for applications in physics to study quantum phenomena and single electron transport and were later also used for solar cells and capacitors [1–3]. However, the potential of using vertical 1D NSs for biological applications has been, and still is, an area gaining increasing attention and interest. The major motivation behind this development is a combination of a size compatibility of cells and NSs, along with the promising scientific, social, and economic possibilities any successful applications could entail. The compatibility of cells and NSs is rooted in the fact that 1D NSs are comparable in length to the size of a cell, and about 100–1000 times smaller in diameter than a cell. Thus, a vertical NS is an object potentially long enough to reach deep into a cell and thin enough to do so without causing damage. Many interesting cellular applications can be envisioned from combining cells and NSs. NSs could deliver [4–6] or collect material from different subcellular compartments and investigate cells otherwise hard to access [7]. The NSs could measure cell composition and activity, either directly owing to their electrical properties [8, 9], through delivery of molecular sensors [10], or by physically probing cellular components [11, 12]. The NSs could also modify the cell activity directly [8, 9], or by delivering a variety of molecules [4–6]. In addition, NSs could be used to modulate [13–15] and even measure [16–18] the adhesion of cells on surfaces.

By up-scaling from a single NS to groups of NSs in random or ordered arrays, one can envision powerful platforms for life science applications. NSs in an array format can allow many cells to be addressed in parallel [8, 9], which can be useful for investigating cell networks or for cell diagnostics. Arrays of NSs can also allow individual cells to be contacted by many NSs simultaneously [8, 9], thereby potentially providing spatial resolution across the cell body. If these multiple contact points across a cell network or cell body also allow interrogation over time [9], the NS array platform would provide temporal resolution in a vast number of cells simultaneously. This would entail gaining unprecedented information on detailed cell activity, which would be highly valuable for the basic understanding of cell signaling and drug discovery. The positioning of the NSs within the array could also be designed to create cellular networks by guiding cells and their processes along the NS pattern [19–21].

Rooted in the very first papers addressing this research subject a decade ago [22, 23], this review is a summary of the intense progress of interfacing cells with NSs that the last few years have brought about. Although both suspensions of NSs [24] and single NSs in field-effect-transistor devices [25] have many interesting biomedical applications, this review will cover arrays of vertical and semivertical high-aspect-ratio NSs for cellular applications. The term 'array' is used in a broad sense to cover groups of NSs with different regularity, i.e., both randomly scattered and orderly arranged NSs. Short NSs with heights below 500 nm or larger structures with widths above 500 nm fall outside the scope of this review. This review covers the broad spectrum of NS materials and dimensions used; the observed cellular responses with specific focus on adhesion, morphology, viability, proliferation, and migration; compares the different approaches used in the field to provide NSs with the often crucial cytosolic access; covers the progress toward biological applications; and finally, the future of this technology is envisioned.

Cell adhesion (figure 3) is mediated by large protein scaffolds known as focal adhesion (FA) points. These FA points are tightly associated with the actin cytoskeleton and together they control a range of cellular responses, such as morphology, migration, and adhesion, that cells use both for sensing and responding to their environment [86].

3.1.1. Adhesion as a function of NS density

3.1.2. Cell position along height of NSs

Cell morphology refers to the basic structure and appearance of a cell. When interfacing cells with NSs, the surface topography plays an important role in dictating the cell morphology (figures 2, 3). Micrometer-sized pillars [72, 89] and minute nanoroughness [15, 74, 75] can drastically alter the morphology of a cell. Cell morphology is a yardstick for normal cell function, but it also has a direct influence on gene expression [90] and cell survival [91], and can thereby affect any aspect of cell health, signaling, and differentiation. Cell morphology is commonly described as a qualitative estimation of cell appearance, but is increasingly being acknowledged for its importance through objective and quantitative investigations.

3.2.1. Qualitative estimation of cell morphology

3.2.2. Quantitative measurement of cell morphology

3.2.3. Morphological maturation of cells

Once a cell has adhered to and spread on the NS surface, it is crucial to investigate the survival of the cells. If the cells become unhealthy, or even die, while interfaced with the surface, the platform would naturally be inadequate for biosensing purposes. The term cell viability (figure 3), or cell health, is used rather unspecifically, as there are many alternative ways to assess it (figure 4). Cell viability is, in many cases, observed indirectly by the mere presence of cells at a certain number of days following interface, assuming that dead cells would have detached by then. Cells have been seen to remain on NS arrays for at least 2–3 weeks [20, 22]. Several properties—such as the status of the cell membrane and nuclear envelope, presence of cellular enzymes, activity of the mitochondria, or synthesis of proteins—can define a healthy cell and therefore, there are many ways to measure the cell viability more directly. Moreover, the assays available can retrieve either ensemble- or single-cell based information, providing an average result for all cells on the sample or details on each individual cell, respectively.

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3.3.1. Integrity of cell membrane and nuclear envelope

3.3.2. Activity of cell enzymes

3.3.3. Activity of mitochondria

3.3.4. Apoptotic cell death

3.3.5. Cell stress and protein expression

The ability of cells to proliferate on NSs is important to consider both as a further measure of cell health, but also with respect to the types of applications the NS array can be used for (figure 3). The term proliferation is often used in a careless manner, through general statements that cells grow, divide, and proliferate on NSs, without providing further data [5, 10, 22, 23]. Both adhesion and viability are to some extent part of the term proliferation, which should more precisely be defined as both cell division and cell number increase. The combination of the two is necessary, as observing a lower relative number of cells over time on NSs, compared to flat controls, does not necessarily correspond to a lower division rate on NSs, as each method on its own do not account for factors such as cell detachment and death.

3.4.1. Cell division

3.4.2. Cell number increase

The migratory pattern (figure 3) has been investigated for primary rat neurons interfaced with an array of ordered platinum NSs. By positioning NSs in a circle, it was demonstrated that the cells remained virtually stationary over a four-day period, while cells on the flat control surface were mobile and migrated up to several hundreds of micrometers during the same period of time [29]. This finding has also been confirmed on both random and ordered arrays of NSs, where cells remain stationary on solid [13, 55] and hollow [6] NSs, whereas they migrate longer distances on flat control surfaces [6, 55]. The reduced migration might be dependent upon surface flexibility, as the cells migrate less on longer NSs rather than on shorter ones [40, 55].

When summarizing all reported cellular responses, it is clear that interfacing with arrays of NSs substantially affects the cellular fate (figure 3). To be able to correlate surface topography with cell responses represents a leap forward for the rational design of ordered arrays of NSs for biological applications. The endeavor is challenging, as the experimental conditions are close to unique for each study in terms of NSs (NS material, shape, diameter, height, array density, regularity, etc), as well as the variability in cell types (table 1) and control surfaces chosen. Interpretations of the results are further complicated, as different conclusions to the same question are drawn based on different types of assays and measurement strategies (figure 4). Furthermore, even with the same experimental setup, assays with different resolutions are seen to provide contradictory results. In a recent study, the single-cell count assay and the ensemble-based Alamar Blue assay—used by others to evaluate the number of cells on a surface [44, 46]—showed opposite results, despite the same combination of NS arrays and cells used [55]. For many of the cell responses it can be very useful to make detailed quantitative measurements, as even small changes of the experimental conditions can have large consequences on the cell fate. In addition, making repeated measurements over time can ensure that the observed response is lasting and is not influenced by the seeding process or the potential cell synchronization following cell interface. The cell responses are more clearly identifiable in systems with well-defined topographies, instead of random arrays where the cell response can vary across the surface. Interestingly though, it seems that several properties can be generalized to a number of systems, i.e., combinations of NS characteristics and cell types. Almost all systems tested support cell adhesion and viability; although, there seems to be a greater risk for cell death and detachment in systems with very high NS density. Cells are generally rounder with a smaller projected cell area on NSs than on flat controls. Although not creating FA points directly on the NSs, it seems cells prefer to adhere to NS arrays, rather than on flat controls, and arrange their adhesion in relation to the NS pattern. Accordingly, cells are captured on NSs and migrate less than on the flat controls.

The many interesting NS-induced cellular responses might be the result of a modified gene and protein expression. This can indirectly be the outcome of remodeled FA signaling complexes [91], possibly in conjunction with adapted levels of intracellular calcium [51], or as a result of the geometric constrains that the NSs introduce to the cells [90, 92]. It can also be a direct effect of the increased NS-induced nuclear membrane curvature, which increases the proximity between chromosomes and the nuclear membrane, and alter the gene expression [93].

NSs were initially believed to gain intracellular access spontaneously when interfaced with cells through standard culture methods [figure 5(a)]. Access was evaluated based on electron microscopy and confocal fluorescence microscopy of NSs excluding a volume of cytosolic dyes [37, 43, 54], as well as DNA expression, although with very low efficiency [43] [figure 5(b)]. This is also the conclusion from a later report, where both the NSs and plasma membrane were fluorescently labelled and imaged with confocal microscopy, showing that most cells were impaled by the NSs within 1 h after seeding [5, 7].

However, experimental evidence eventually suggested that spontaneous access is not always gained, possibly only for as few as 1–10% of the NSs [6, 68]. With a detailed single-NS resolution, we used fluorescence labeling of a membrane protein [figures 3, 5(b)] to show that the plasma membrane generally enclosing the NSs, irrespective of the wide range of NS heights and densities investigated [57]. The method developed was used by Mumm et al on a platform with other types of NSs and cells, confirming that the NSs remain wrapped in cell membrane also under these conditions [59]. Nevertheless, highly efficient intracellular delivery of small fluorescent or nonfluorescent molecules is still reported with both solid [5, 7, 52, 59] and hollow [6] NSs [figure 5(b)]. A high delivery efficiency, however, is likewise observed on flat control surfaces [52, 59], indicating that delivery of fluorescent molecules might not be directly associated with NSs penetrating the cell membrane, but rather assisted by the local up-concentration of molecules at the NS-cell interface, or by defects at the highly curved membrane that are too small or transient to be detected with a membrane labeling technique. Either way, this stresses the importance of including proper controls when using the delivery of molecules to evaluate intracellular access. Delivery of functional siRNA or DNA is also reported, but with generally lower efficiencies than for the previously mentioned small molecules. This shows how the delivery of certain molecules does not necessarily equate delivery of especially larger molecules or molecules that require maintenance of functionality. Furthermore, several studies using transmission [59, 63] and scanning electron microscopy following focused ion-beam milling [9, 48, 55] have investigated the interface between NSs and the cell membrane. These techniques also reveal that the plasma membrane wraps tightly around the NSs [9, 59, 63] (figure 3). Using a recently developed theoretical model, we actually predict this behavior, because in many cases during standard culture, the cells can energetically afford to simply bend the membrane around the NSs [13], giving them no incentive to be penetrated. However, a very recent theoretical study proposes a post-deformation, adhesion-mediated mechanism for spontaneous penetration and explores the probability of such intracellular access as a function of NS array dimensions and mechanical properties of the cells [94].

As intracellular access is not spontaneously acquired for many platforms, alternative strategies are developed to obtain intracellular access and to further increase the number of NSs gaining access [figure 5(a)]. One strategy evaluated is to modify the surface coating of the NSs. By increasing the nitrogen content of vertically aligned carbon nanofibers, they are able to retain more DNA, and subsequent interface with cells was shown to yield a higher number of DNA-expressing cells [22]. Interestingly, modifying the NSs with aminosilane increases the number of NSs gaining a direct intracellular access from 0–7% [57] compared to bare and polyethyleneimine-coated NSs [figure 5(b)]. Furthermore, by coating NSs with other adhesion promoters, the Melosh group recently demonstrated that polyorthinone and fibronectin both increase the number of NSs with cytosolic access, as well as significantly affecting the penetration kinetics, thus making the direct cytosolic access a quicker and more likely event [68]. The same group has also developed an approach to maintain the direct access of NSs to the intracellular compartment, where the addition of a hydrophobic ring around the NS increases the adhesion strength to an artificial bilayer [95, 96] or to the membrane of a red blood cell [97]. The relevance of defining a hydrophobic region to assist membrane penetration has also been confirmed by modifying other types of NSs with a phospholipid bilayer [98].

Applying force to the cells during interfacing is one of the earliest tactics applied to enhance intracellular access [22, 23, 30, 32, 33] [figure 5(a)]. It has been shown that centrifuging cells onto carbon NSs is not enough for efficient delivery of DNA. Inverting the NS array on top of the cells and subsequently pressing or tapping it with tweezers somewhat improves the delivery efficiency but also introduce large variations in delivery efficiency across each array [22, 23, 32, 33] [figure 5(b)]. In an interesting study, outside the scope of this review, the force needed to penetrate the plasma membrane was probed using a single NS on an atomic force microscope tip; subsequently, the short-term viability of the penetrated cells was also investigated [99–101].

A recent strategy that to some extent also includes forcible penetration has been denominated the 'sandwich' assay. Here, the cells adhere to one NS array that is inverted and placed on top of another NS array (the probe array) functionalized with enzyme substrates. Intracellular access is either shown by a fluorophore on the substrate, which is then cleaved with intracellular enzymes and left behind in the cytosol, or by examining the probe array using mass spectrometry to verify substrate cleavage, thus showing retroactively that the substrate has been in contact with intracellular enzymes [10] [figure 5(b)].

Detergents are commonly used in immunocytochemistry to deliver the large antibodies across the membrane of fixed cells. By using a mild detergent solution to destabilize the membrane [figure 5(a)], Peer et al show that macromolecules are able to enter the cell through hollow NSs, while still maintaining cell viability [4]. The direct intracellular access of the NSs is shown through delivery of a fluorescently labelled molecule and a DNA plasmid encoding a fluorescent protein from a reservoir below the hollow NSs. To achieve delivery, it is necessary to both have NSs present (pores without NSs do not deliver cargo) and a low concentration of a detergent in the solution. This demonstrates that a combination of mechanical strain and detergent can facilitate the intracellular access of NSs.

The cell membrane can likewise be destabilized using electroporation [figure 5(a)]. Nanoelectrodes are small, but tightly coupled to the membrane, and they can thus create a strong electric field using only a small voltage to increase the cell membrane permeability transiently and locally. Using such NS-based electroporation, direct intracellular access of NSs has been demonstrated by several groups. The shape of the measured action potentials shows when access is gained and it is demonstrated that NSs can gain access to the same cell repetitively over several days [9, 70]. In a different system, access is spontaneously gained to more than half of the cells, and access can be obtained for the rest through electroporation [8]. In a platform combining electroporation and relatively thick (250 nm diameter) hollow NSs to avoid any spontaneous penetration, Xie et al show that electroporation is necessary to obtain intracellular access of the NSs. The access is similar to previous reports shown by delivery of a fluorescent dye and DNA plasmids encoding fluorescent proteins through the hollow NSs with high efficiency [67] [figures 3, 5(b)]. Intriguingly, all of these electrophysiology-based systems [8, 9, 67] congruently reveal that the intracellular access of the NSs is transient, on the order of minutes, before the membrane reseals.

In conclusion, a variety of methods has been used to interface cells with NSs [figure 5(a)] that, with variable success rates, have assisted the intracellular access of NSs. Modifying the NS surface coating increases the number of NSs gaining direct intracellular access by up to seven percentage points [57, 68]; by applying force, the number of cells expressing delivered DNA increases to at least 5% [23]; both detergent solutions and electroporation provided high efficiencies of molecule delivery (70–95% [4, 67]) and DNA expression (70–80% [67]), as well as a major improvement in the number of NSs with direct electrophysiological access to the intracellular compartment [8, 9] [figure 5(b)]. As previously shown, different conclusions are drawn regarding the ability of NSs to spontaneously gain intracellular access [figure 5(b)], which can have several explanations. Naturally, the NS access may differ when different cell lines are presented with the same topography, or when one single-cell line is presented with different topographies. Therefore, the dimensions and positioning of the NSs should ideally be controlled to promote consistency between experiments. Furthermore, even though there is a significant difference between the delivery efficiency measured per NS or per cell, i.e., the intracellular access of NSs versus molecule delivery efficiency, the two concepts are commonly made equivalent. Thus, when discussing intracellular access of NSs, one should remember that the situation might not be identical for all NSs in a given sample. It is likely that any platform and interfacing method will result in some NSs gaining intracellular access, whereas others are wrapped by the plasma membrane. The method used to evaluate intracellular access may or may not allow estimation of the share of NSs that gain access. Some methods, such as membrane labeling and electrophysiology, can offer single-NS resolution, whereas others, such as molecule delivery, offer only single-cell or even ensemble-based resolution [figure 5(b)]. Finally, as illustrated by the electrophysiology-based systems, NS access can be transient and vary over time, thus also making the time point of observation important to consider.

Cell membranes represent formidable barriers for delivery of exogenous macromolecules, which allows cells to maintain their integrity. To overcome this barrier is a major challenge in biological and medical research, where it is of great interest to deliver a broad diversity of molecules to study basic cell physiology and pathology and develop drugs to counteract disease. Traditional molecule delivery methods such as electroporation, viral vectors, and standard transfection reagents can be detrimental or toxic to cells, yield inconsistent transfection efficiencies [102], and generally require large volumes of precious biomolecules. In many standard cell lines, decent transfection efficiency can be achieved using standard transfection reagents. Although cells can be efficiently transfected with standard methods while in contact with NSs [53], using the NSs per se for this purpose can be a useful alternative. When using NSs for delivery [figure 6(a)], it has been demonstrated that these transfection efficiencies can be matched or even surpassed [figure 5(b)]. Several fluorescent and nonfluorescent molecules, such as siRNA, peptides, proteins, and DNA sequences have been delivered into cells with efficiencies ranging from 40–99% [5–7, 52, 59, 68]. The delivered molecules sometimes appear to remain enclosed in cytosolic vesicles though, which could explain why the subsequent efficiency of DNA expression often is well below 1% [22, 43, 59], and rarely up to 5–10% [6, 23] [figure 5(b)]. High expression efficiencies (70–80%) are thus far only achieved using a combination of hollow NSs and electroporation [67]. The role of the NSs in small-molecule delivery has been questioned, as recent evidence shows that high delivery efficiencies are, in some cases, also attained by the flat controls, in the absence of NSs [52, 59]. Encouragingly though, Chan et al demonstrated that although both NSs and flat control surfaces provide high delivery efficiencies, only by using the NS arrays would the delivered cargo avoid ending up in intracellular endo- and lysosomes [52].

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Other cell types, such as certain primary cells, resting cells, suspension cells, and stem cells, have proven considerably harder to transfect [103, 104]. These cells are highly interesting in cell biology, but present an additional challenge, as they are resistant to even optimized transfection methods and either remain nontransfected or die during the process. Here, NSs have proven useful as promoters of exogenous molecule delivery into different types of primary immune cells.

A drawback of traditional transfection methods is that they generally do not allow for continuous or sequential delivery of molecules into the same cells. By using hollow NSs connected to an underlying reservoir instead [6], molecules can be delivered repeatedly into more than 70% of cells through passive diffusion in a detergent solution [4]. In a similar approach, hollow NSs were combined with localized electroporation, which demonstrated an on-demand, repeatable delivery to more than 95% of the cells [67]. The latter approach also demonstrated the highest NS-based transfection efficiency noted thus far, where the delivered DNA plasmids were expressed in up to 80% of the cells. These approaches are superior to microinjections, as they are done in an easy, high-throughput manner with benign manipulation of many cells simultaneously.

In addition to potentially modifying cell activity, molecules for detecting cell activity could also be delivered into cells [figure 6(b)]. The group of Nakamura is a major player in this field, where single NSs are used to measure enzymatic activity [105], deliver DNA molecules [106, 107], and investigate the status of the cell cytoskeleton [11, 12]. Recently it was demonstrated that this concept could be up-scaled to an array format, where the intracellular activity of three different enzymes was investigated. The enzymatic substrates were bound to an NS array, which was introduced into cells. After retraction of the NS array, the enzyme activity was evaluated based on intracellular fluorescence and mass spectrometry of the NSs [10]. This is an optimistic development toward real-time detection of cellular activity using NS arrays.

The most common method for obtaining an electrophysiology-based readout of cell activity is using micrometer-sized patch-clamp probes. This technique is generally laborious, low-throughput, stressful to cells, and suffers from limited recording times [108, 109]. To upscale the method, micron-sized mushroom-like probes were demonstrated to enable intracellular-like neuronal measurements in an array format [110]. This work was soon followed by two exceptional demonstrations of intracellular label-free recordings of cell activity based on arrays of vertical NSs [8, 9] [figure 6(c)]. In these studies, pads of positioned vertical NSs were used to electroporate and measure the electrophysiological activity of cells. It was demonstrated that NS-mediated current injections could evoke action potentials in primary neurons [8], and that the action potential activity could be followed in the same cells over an unprecedented period of several days [9]. Further support for the use of NSs for electrophysiological purposes is that, as the membrane seals tightly around the NS, megaohm [8] to gigaohm [97] seal resistances are generated, which thereby greatly minimize the signal loss for NS-based electrodes. Recently, it was demonstrated that by using hollow instead of solid NSs, the cell-electrode coupling improved the signal even further and allowed for much longer recordings [70].

Arrays of vertical NSs are also developed for applications where the intracellular access of NSs is not imperative. The mere presence of NSs can physically influence the cells and thereby guide cells to form a network [figure 6(d)]. This has been demonstrated for neuronal cells, that either followed on top of [20, 21] the NS pattern or in between the NSs [19], thus creating artificial neuronal networks. The material, height, and diameter provide the NSs with a certain flexibility, and the forces exerted by cells have been measured by quantifying the deflection of individual NSs [16–18]. As described earlier, cells remain close to stationary on NSs [29, 55], providing the opportunity to use NS arrays to capture cells and to follow the same cell for extended periods of time. Additionally, because arrays of NSs influence the developmental fate of cells and allow growth of many specialized cell types, NS arrays are also considered for uses in tissue engineering and regenerative medicine [14, 38, 44, 50, 64, 111] and for their appropriateness as implants [27, 62]. Arrays of vertical NSs have moreover been proven useful for acellular applications, such as protein arrays [112, 113].