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. Author manuscript; available in PMC: 2012 Mar 1.
Published in final edited form as: Biochim Biophys Acta. 2010 May 24;1810(3):330–338. doi: 10.1016/j.bbagen.2010.05.005

Bio-functionalized nanoneedles for the direct and site-selective delivery of probes into living cells

Kyungsuk Yum, Min-Feng Yu, Ning Wang, Yang K Xiang *,#
PMCID: PMC2948073  NIHMSID: NIHMS214994  PMID: 20580773

Abstract

Background

Accessing the interior of live cells with minimal intrusiveness for visualizing, probing and interrogating biological processes has been the ultimate goal of much of the biological experimental development.

Scope of Review

The recent development and use of the bio-functionalized nanoneedles for local and spatially-controlled intracellular delivery brings in exciting new opportunities in accessing the interior of living cells. Here we review the technical aspect of this relatively new intracellular delivery method and the related demonstrations and studies, and provide our perspectives on the potential wide applications of this new nanotechnology-based tool in the biological field, especially on its use for high resolution studies of biological processes in living cells.

Major Conclusions

Different from the traditional micropipette-based needles for intracellular injection, a nanoneedle deploys a sub-100 nm diameter solid nanowire as a needle to penetrate a cell membrane and to transfer and deliver the biological cargo conjugated onto its surface to the target regions inside a cell. Although the traditional micropipette-based needles can be more efficient in delivery biological cargoes, a nanoneedle-based delivery system offers an efficient introduction of biomolecules into living cells with high spatiotemporal resolution but minimal intrusion and damage. It offers a potential solution to quantitatively address biological processes at the nanoscale.

General significance

The nanoneedle-based cell delivery system provides new possibilities for efficient, specific, and precise introduction of biomolecules into living cells for high resolution studies of biological processes, and it has potential application in addressing broad biological questions.

1. Introduction

Nanotechnology has recently found increasing applications in biology by providing new nanotechnology-based tools and materials to probe and manipulate biological processes at the nanoscale (~1 to 100 nm) [1], which is the length scale where many fundamental biological processes occur. For instance, fluorescent semiconductor nanoparticles, or quantum dots [2, 3], have been used as probes to visualize dynamic processes in living cells, including the dynamic movement of singe membrane receptors [4-8], motor proteins [9], nerve growth factors [10], and synaptic vesicles [11, 12]; and magnetic nanoparticles have been used to manipulate individual membrane receptors to control signal transduction in living cells [13].

One-dimensional nanomaterials, such as nanotubes and nanowires, have also been used as intracellular biosensors, delivery carriers, and imaging agents [14-21]. In particular, with their unique physical and chemical properties distinct from both individual molecules and bulk materials, chemically synthesized nanomaterials have presented new opportunities and applications in biology and medicine, from basic biophysical studies at the single-molecule level to the diagnosis and treatment of diseases [22-24]. Additionally, with their needle-like nanoscale geometry and excellent mechanical and electrical properties, these high-aspect ratio nanostructures have been explored as membrane-penetrating nanoneedles that can manipulate and sense biological processes inside cell with minimal intrusiveness and toxicity [24-31]. For example, surface-functionalized nanotubes have been used to deliver biomolecular species into living cells with high spatial and temporal precision [27, 30, 31]. Conductive nanotubes have also been envisioned as an electrochemical nanoprobe to measures electrochemical events, redox environments, and signaling processes occurring inside cells or between neighboring cells [31, 32].

The transfer of biomolecules into living cells is a general practice used to monitor or modify molecule-specific intracellular processes. It provides an efficient way to study the temporal and spatial regulation of protein systems that underlie basic cellular functions [33]. Many methods have been developed for this purpose [33-41]. Each of them has its characteristic advantages and disadvantages with respect to cell viability, transfer efficiency, general applicability, and technical requirements [33]. In this review, we discuss a new type of nanotechnology-based methodology for the introduction of biomolecules into living cells and its potential implications in addressing biological questions.

2. General Description of the Nanoneedle-Based Intracellular Delivery

Similar to a micropipette-based injection system, a nanoneedle-based intracellular delivery system comprises a nanoneedle (a nanotube or a nanowire) on a macroscopic handle (an etched metallic wire or simply a pulled glass micropipette) and a manipulator (a standard piezoelectric micromanipulator) integrated with an inverted optical microscope [26, 27, 30, 31]. Similar also in practice to the use of a standard micropipette-based injection system, the nanoneedle is manipulated with the micromanipulator to penetrate into a target cell under the observation of an optical microscope. The major difference between the nanoneedle-based system and the micropipette-based injection system is that in the nanoneedle-based system a sub-100 nm diameter nanowire is used (compared to the micrometer-sized micropipette used in the injection system) to penetrate the cell membrane, which introduces minimal damage to the cell membrane and minimal disruption to the interior environment of a cell; and the materials to be delivered into the cell are carried by the nanoneedle surface and released through a predesigned surface chemistry [27, 30, 42-44] and not through a pressure-drive injection flow.

Such a configuration also allows the direct visual monitoring of the whole nanoneedle-based delivery process (Fig 1A) and requires no additional setup beyond what a biological science laboratory typical has. The drawback is that its operation is limited by the resolution of the optical microscope; thus, only nanoneedles with relatively large diameter and length (diameter larger than ~30 nm and length larger than ~3 μm) can be visually monitored and thus used. Other configurations have used a nanoneedle mounted on an atomic force microscope (AFM) probe and manipulated by an AFM. The advantage of such an AFM-based nanoneedle system is that the force-displacement dependence behavior when the nanoneedle approaches towards and breaks through the cell membrane, and advances into the cell interior can be quantitatively monitored with very high resolution (Fig. 1B, C) [26-28]. However, the limitation is that the direct visualization of the nanoneedle is difficult as the AFM cantilever blocks the direct view of the nanoneedle, and the nanoneedle motion is restricted along the vertical direction. Some AFM-based systems have also been integrated with a confocal microscope; this allows the three-dimensional imaging of the nanoneedle and the target cell (Fig. 1B, C) [26, 43]. However, because of the long acquisition time needed in confocal imaging, real-time monitoring of the nanoneedle operation and the dynamic cellular processes is difficult [45].

Fig. 1.

Fig. 1

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Nanoneedle manipulation with a micromanipulator and an AFM system. (A) Optical microscope images of a nanoneedle functionalized with quantum dots when the nanoneedle was manipulated by a micromanipulator to penetrate into the cytoplasm of a living HeLa cell. The whole process was monitored in situ in the bright field (left), the fluorescence (middle), or the combined bright-field and fluorescence (right) image mode. The quantum dots attached on the nanoneedle are shown in red (middle) and bright white (right). The unfocused dark shade on the right side is the tip of the macroscopic needle on which the nanoneedle is attached. (B) Cross-sectional laser-scanning confocal microscope images of a living human mesenchymal stem cell (MSC) expressing red-fluorescent protein (DsRed2-NES) (top) and a Si nanoneedle conjugated with fluorescein isothiocyanate (FITC) with green emission when the nanoneedle was manipulated by an AFM system to penetrate into the nucleus of the MSC cell (middle), and a force-distance curve obtained from the AFM during the nanoneedle operation (bottom). (C) The same observation for a human embryonic kidney cell (HEK293). The nanoneedle contacted the cell membrane (point I), began to feel the repulsive force as it indented the cell (point II), penetrated though the cell membrane (point III), and contacted the substrate (point IV). (A) from Ref. [30] (Copyright 2009 American Chemical Society), (B) and (C) reproduced with permission from Ref. [43] (Copyright 2008 Elsevier).

Overall, an ideal nanoneedle-based delivery system that can control the nanoneedle at the nanoscale resolution, directly visualize the nanoneedle and the target cell, and monitor the force exerted on the nanoneedle would be desirable for the wider use of nanoneedles for biological studies in living cells [45, 46].

In the following, we discuss the fabrication and functionalization of a typical nanoneedle for the intracellular delivery application.

3. Fabrication of Nanoneedles for Intracellular delivery

The most critical component in the nanoneedle-based system is the nanoneedle, which is often attached to a macroscopic structure for the ease of manual handling. For the intracellular delivery purpose, the nanoneedle, in general, needs to have a needle-like structure with nanoscale diameter (less than ~100 nm) and microscale length (~1 to 20 μm, long enough to reach the target area inside a cell), be mechanically rigid to survive the operation in an aqueous media and to penetrate through the cell membrane [25, 28], and have a surface that can be chemically functionalized to attach the cargo on its surface or to convey other functionalities to the nanoneedle [28].

Such a nanoneedle can be typically made by the following two methods. First, chemically-synthesized one-dimensional nanostructures, such as nanotubes and nanowires, can be directly used as nanoneedles [27, 28, 30]. For example, chemically-synthesized nanotubes (carbon nanotubes or boron nitride nanotubes) have ideal properties as nanoneedles for intracellular delivery: they have the needle-like structure with nanoscale diameter (~1 to 100 nm) and microscale length (~1 to 100 μm) [47], large Young's modulus (~1 TPa) and high tensile strength [48-51], while in the meantime are resilient [48, 49, 52, 53]. There are well-developed methods to synthesize such one-dimensional nanostructures with controlled sizes and shapes and they are mostly commercially available. However, the precise alignment and stable assembly of such nanostructures into useful individual nanoneedles are still challenging [25]. Reported methods for the assembly of the nanostructures into needle-like structures include direct attachment of nanotubes by using a manipulator [27, 28, 30, 52-55], catalyst-patterning and direct growth of nanotubes by chemical vapor deposition [56-58], alignment and assembly using dielectrophoresis or magnetic fields [25, 59-66], and transplanting of single nanotubes encapsulated in polymer blocks [67, 68]. However, these methods do not reproducibly produce nanoneedles in large quantity or produce high-aspect ratio “water-stable” nanoneedles (that can survive the meniscus forces involved in the intracellular delivery operation) [25, 28, 54]. Second, a nanoneedle can be fabricated by nanofabrication, such as focused ion beam (FIB) machining [26] and direct-write nanofabrication techniques [69-71]. For example, Nakamura and colleagues have fabricated Si nanoneedles by sharpening Si AFM tips with FIB [26, 42-44, 72-76]. They fabricated Si nanoneedles with diameters of ~200 to 800 nm and lengths of ~5 to 10 μm and demonstrated the capability of these Si nanoneedles to penetrate through both the cellular and nuclear membranes of living cells. However, in general, nanofabrication methods produce nanoneedles with relatively large diameters (in most cases, larger than 100 nm) and short lengths.

4. Functionalization of Nanoneedles for Intracellular Delivery

Several research groups have developed the nanoneedle-based delivery system that uses the outer surface of the nanoneedle for carrying the cargo for intracellular delivery (Fig. 2) [27, 30, 42-44]. This requires that the cargo is conjugated on the surface of the nanoneedle and is able to be released from the surface of the nanoneedle once transferred inside cells. There are various surface chemistry and bioconjugation methods to functionalize the surface of the nanoneedle and conjugate the cargo on it. For example, the surface of the nanoneedle can be directly functionalized (Fig. 2A) [27, 43, 66] or can be first coated with other materials (e.g., gold) and then functionalized (Fig. 2B) [28, 30].

Fig. 2.

Fig. 2

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Surface functionalization of nanoneedles and loading of cargo. (A) Surface functionalization of a CNT nanoneedle with streptavidin-conjugated quantum dots (QDs) through a linker molecule containing a disulfide bond. (B) Surface functionalization of a gold-coated nanoneedle with streptavidin-conjugated quantum dots (QDs) through a stepwise procedure. (A) reproduced with permission from Ref. [27] (Copyright 2007 National Academy of Sciences, U.S.A.), (B) from Ref. [30] (Copyright 2009 American Chemical Society).

In the case of a carbon nanotube-based nanoneedle, it can be functionalized by either covalent methods or non-covalent methods. The covalent methods use chemical reactions to chemically bond functional groups directly on the surface of the CNT (e.g., carboxyl groups by oxidation) [20, 21, 66, 77]. The non-covalent methods use hydrophobic and π-π interactions [20, 21, 27, 77] to attach functional groups on the CNT surface. For example, Chen et al. [27] used a linker molecule that contains a pyrene moiety and a biotin moiety to functionalize the CNT nanoneedle with nanoparticles: the pyrene moiety binds to the CNT surface through π-π stacking and the streptavidin-coated nanoparticles are attached to the biotin moiety (Fig. 2A).

A Si nanowire-based nanoneedle can be functionalized by forming self-assembly monolayers (SAMs) of alkylsilanes on the Si surface through the silane coupling reaction. For instance, Nakamura and colleagues used (3-aminopropyl)triethoxysilane and (3-mercaptopropyl)trimethoxysilane to functionalize the surface of Si nanoneedles with SAMs with amine and thiol terminal groups and used these functional groups to conjugate proteins, DNA, and polymers [42-44, 73, 75].

A more general approach is to coat the nanoneedle with a common material, such as gold, and subsequently functionalize the coated nanoneedle [28, 30]. This approach is nonspecific to the type of nanoneedles; thus, a “standard” surface chemistry method can be used for various nanoneedles rather than synthesizing linker molecules for a specific type of nanoneedles. The surface coating also increases the mechanical integrity of the nanoneedle structure [28, 30, 78]. A well-studied method is to coat the nanoneedle with a thin layer of gold (~1 to 20 nm in thickness) and form a SAM through the chemisorption of thiols on gold surfaces [28, 30]. For example, Yu and colleagues [30] developed a stepwise procedure to functionalize gold-coated nanotube nanoneedles: they first formed an amine-terminated SAM on the gold-coated nanoneedle, then conjugated a linker molecule containing a disulfide bond within its spacer and a biotin moiety, and finally attached streptavidin-coated nanoparticles on the biotinylated nanoneedle by the binding of streptavidin and biotin (Fig. 2B). Moudgil and colleagues [28] also conjugated gold nanoparticles on gold coated CNT nanoneedles by forming an amine-terminated SAM and subsequently attaching gold nanoparticles by electrostatic interactions.

Comparing to the ready availability of numerous surface functionalization methods for conjugating various materials on the nanoneedle, a more challenging task is to design a conjugation strategy that can allow the controlled release of the conjugated materials from the nanoneedle once transferred inside cells (Fig. 3). A simple approach is to rely on the passive desorption of the non-chemically attached cargo from the nanoneedle. For example, Nakamura and colleagues [42-44] attached DNA on the functionalized Si nanoneedle (~200 nm in diameter) through electrostatic interactions and demonstrated the release of the DNA from the nanoneedle through the passive desorption of DNA inside cells (Fig 3A). They demonstrated the delivery of plasmid DNA into the nucleus of single human mesenchymal stem cells (MSCs), human embryonic kidney cells (HEK293), and breast cancer cells (MCF-7), using an AFM-based nanoneedle system. In particular, the direct delivery of green fluorescent protein (GFP) plasmid DNA into the nucleus of single target cells not only selectively expressed GFP in the single target cells but also expressed GFP at a high successful rate (~70% for primary cultured human MSCs), compared favorably with other nonviral gene delivery methods (lipofection ~50% and microinjection ~10% for human MSCs) (Fig 4A) [43]. However, because it is nonspecifically adsorbed on the nanoneedle, the cargo (i.e., DNA in the study) is unselectively desorbed from the nanoneedle both in the media and inside the target cell in this approach. Thus, the delivery process must be completed within several minutes after inserting the nanoneedle into the cell media (before the complete desorption of cargo from the nanoneedle into the media).

Fig. 3.

Fig. 3

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Schematics of nanoneedle-based intracellular delivery. (A) Gene delivery using a DNA-adsorbed Si nanoneedle. The DAN is passively desorbed from the nanoneedle in the nucleus of the cell. (B) Intracellular delivery though the reductive cleavage of the disulfide bond. The nanoneedle functionalized with cargo via a disulfide bond penetrates the cell membrane. The cargo is released from the nanoneedle through the reduction of the disulfide bond in the cytosol of the cell. The nanoneedle is then retracted. (A) reproduced with permission from Ref. [43] (Copyright 2008 Elsevier), (B) from Ref. [27] (Copyright 2007 National Academy of Sciences, U.S.A.)

Fig. 4.

Fig. 4

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Nanoneedle-based delivery of molecules into the cytoplasm and nucleus of living cells. (A) GFP expression in a mesenchymal stem cell (MSC) using a DNA-adsorbed nanoneedle (shown in Fig. 3A): bright-field (left) and fluorescence (right) images of the MSC (24 hours after delivery). (B) Delivery of quantum dots into the cytoplasm of a HeLa cell using the nanoneedle-based mechanochemical delivery method: bright field (left), fluorescence (middle) images of the cell after delivery and overlay of the bright field and fluorescence images (right). (C) Delivery into the nucleus of a HeLa cell using the same method in (B): overlay of bright-field and fluorescence images of a cell after delivery (left) and enlarged fluorescence image (middle) and overlay of bright-field and fluorescence images (right) of the marked region in the left image. The arrows indicate quantum dots (red). The dotted line locates the boundary of the nucleus. (A) reproduced with permission from Ref. [43] (Copyright 2008 Elsevier), (B) and (C) from Ref. [30] (Copyright 2009 American Chemical Society).

A more sophisticated method to release cargo specifically inside cells is to use the reductive cleavage of the disulfide bond in the reducing environment of the interior of cells; cells have the regulatory mechanism that maintains the redox equilibrium of the intracellular environment, where the disulfide bond is reductively cleaved into two thiol groups (Fig. 3B) [27, 30]. Using this strategy, Bertozzi and colleagues [27] and Yu and colleagues [30] demonstrated the delivery of a discrete, small number of protein-coated fluorescent quantum dots into living HeLa cells: they attached the quantum dots on the nanoneedle through a linker molecule containing a disulfide bond (Fig. 2B) and demonstrated the release of the quantum dots inside cells by incubating the nanoneedle inside cells for ~15 to 30 minutes (a time required for the reduction of the disulfide bond) (Fig. 3B). Furthermore, Yu and colleagues [30] demonstrated the selective delivery of well-dispersed single quantum dots into the cytoplasm and nucleus and the tracking of the delivered quantum dots inside the cells (Fig. 4 and Fig. 5).

Fig. 5.

Fig. 5

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Tracking of quantum dots delivered into living HeLa cells. (A) A series of fluorescence images showing the trajectory (the green line) of a tracked QD. (B) Mean-square displacement (MSD) versus time data showing three types of characteristic motion of QDs: free diffusive (red square), confined (blue square), and stationary (black square). The solid red line and blue line are the line fit on the data based on a free diffusion model and a confined diffusion model, respectively. (C) Diffusion coefficient of QDs in the cytoplasm: the diffusion coefficient D (0.08−3.8 μm2/s, mean 0.8 μm2/s, n = 20) was determined by fitting a free diffusion model MSD(t) = 4Dt to the initial few data points of each MSD versus time curve acquired from tracking of different QDs. The solid red lines are the fitted line. The dashed blue line indicates the MSD expected for freely diffusing QDs in aqueous solution (D0 = 17 μm2/s). The dashed green lines are reference lines for D of 1.0 and 4.0 μm2/s. Reproduced from Ref. [30] (Copyright 2009 American Chemical Society).

5. Features of the Nanoneedle-Based Intracellular Delivery

The intracellular delivery using a cell membrane-penetrating nanoneedle has several unique features that may allow new strategies for biological experiments inside living cells. The nanoneedle can deliver a discrete, small number of molecules into a target area of a single target cell at desirable times without any apparent damage to the cell. The general advantages and disadvantages of the nanoneedle-based delivery method in comparison to other common delivery methods are summarized in the Table 1 [33].

Table 1.

Common Cellular Delivery methods

Method Target cell Cargo Efficiency Cell survival Quantitative Automation Others
Nanoneedle-based delivery Unlimited Diversified High (up to 100%) High Great No Expensive and technically
demanding, limited number of
cells, spatial precision in
delivery
Microinjection [33, 34] Unlimited Diversified High (100%) Fair Fair No Expensive and technically
demanding, limited number of
cells
Viral infection [31] Unlimited DNA only High High Poor Yes Virus packing and viral
gene expression
Electroporation [35] Limited Limited Fair Fair Poor No Requires specialized equipment
works best when cells are in
suspension
Protein transduction [32] Unlimited Diversified Fair High Poor Yes Receptor independent
Cargo Size restriction,
in vivo applications possible
Protein unfolding
Liposome-mediated transfer [36, 37] Limited Diversified Fair Fair Poor Yes Interferes with lipid metabolism
in vivo applications possible
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Table 1 is adapted from Ref. 30.

The nanoneedle can deliver cargo into living cells with spatial and temporal precision. The ability of the nanoneedle to reach target areas inside cells allows the direct delivery of cargo into target areas or compartments inside cells, not readily achievable by conventional delivery methods. For instance, it is demonstrated that the nanoneedle-based method can selectively deliver quantum dots into either the cytoplasm or the nucleus of living HeLa cells (Fig. 4B, C) [30]. This capability may potentially allow spatially-resolved experiments inside living cells (e.g., inside the nucleus). Because the cargo is released from the nanoneedle inside cells, the spatial resolution of the delivery is mostly determined by the size of the nanoneedle inserted into a cell (< ~100 nm in diameter and ~1 to 4 μm in length) and the spatial precision of the manipulator (e.g., a nanoscale resolution of ~1 nm is achievable with a piezoelectric manipulator, such as an AFM system). The delivery can also be done with high temporal precision (e.g., at desirable times throughout the cell cycle); the temporal resolution of ~15 to 30 minutes is achievable, for example, when the reductive cleavage of the disulfide bond is used as a release mechanism (Fig. 3B). Currently, we are also working on an electrochemical delivery approach, in which the release of the attached cargo can be achieved by applying a pulse of a small electrical potential to the nanoneedle. Such a delivery strategy can significantly enhance the temporal resolution down to the several seconds range. The delivery with such a high temporal precision will allow the manipulation of cellular processes with short time scales, such as signaling transduction and protein transportation inside of cells.

The nanoneedle can deliver a discrete, small number of molecules into cells. For example, the nanoneedle-based method can uniquely deliver well-dispersed single quantum dots into cells [30]; this capability may potentially allow the use of the delivered quantum dots for molecular imaging inside living cells (Fig. 4B, C). Despite their bright and stable fluorescence, ideal for single-molecule imaging, the lack of methods that can deliver singly-dispersed quantum dots into cells has hindered their use for molecular imaging inside living cells, one of the most promising applications of quantum dots [2, 6]. The unique capability of the nanoneedle to precisely deliver only a small number of nanoprobes can minimize the interference of the delivered nanoprobes (e.g., quantum dots and magnetic nanoparticles) with intended experiments in living cells and the effect of such nanoprobes on cellular physiology. For instance, the delivery of a small number of quantum dots significantly reduced the background signal from the out-of-focus quantum dots, enabling the detection and tracking of single quantum dots inside living cells even with a simple epifluorescence microscope (Fig. 5A) [30]. The direct tracking showed that the quantum dots diffused in the cytoplasm of HeLa cells with varying diffusion coefficient D of ~0.1 to 4 μm2/s, indicating the high heterogeneity of physical properties and the molecular crowding of the intracellular environment (Fig. 5B, C) [30]. In contrast, a recent study also showed that endosomal accumulation of quantum dots, introduced into cells via the endocytic pathways, can have several effects on cell physiology [79]. Additionally, spatially-resolved delivery of one or a traceable number of force probes (e.g., magnetic nanoparticles) would be desirable for some cellular and molecular mechanics experiments inside living cells, in which one needs to know where the force is applied [80].

How the nanoneedle affects cellular function and viability is important in any living cell experiments. Most studies showed that the penetration of a nanoneedle into a living cell does not impair the cell viability or membrane integrity. For example, the cell viability tests, using the trypan blue exclusion assay, the Calcein AM assay, and the Annexin V-FITC/propidium iodide assay, or the monitoring of the cell proliferation with nanoneedles with diameter less than 100 nm showed that mammalian cells, including HeLa cells, mouse embryonic stem cells and human embryonic kidney cells (HEK 293), were viable for the duration of the experiments (from several minutes to several days) [27, 29, 30]. Cell viability tested with nanoneedles with different diameters, using the 4,6-diamido-2-phenylindole (DAPI) exclusion assay for human epidermal melanocytes, HEK 293, and breast cancer cells (MCF-7), also suggested that maintaining nanoneedles with diameter less than 400 nm inside cells does not affect the cell viability for at least one hour [42]. However, how the nanoneedle affects the cellular processes and functions needs more thorough examination.

6. Biological Applications of Nanoneedle-Based Intracellular Delivery

While it is exciting to have a functionalized nanoneedle as a new gene/ protein delivery system for cell studies, more remains to be explored to utilize such an elegant yet powerful system for studying different cellular processes. In this section, we highlight a few preliminary attempts and potential applications with the functionalized nanoneedle for intracellular delivery.

Delivery of DNA/RNA molecules for gene expression: high efficiency in gene delivery remains highly desirable for cellular studies; in particular, high efficiency delivery of exogenous genes into many primary cultures as well as stem cells remains a challenge. As mentioned previously, Nakarmura and colleagues [42-44] have demonstrated the direct delivery of DNA into the nucleus of a variety of mammalian cells, including human mensenchymal stem cells (Fig 4A). With improvement on the releasing methods, it is expected that the direct delivery of plasmid DNA into the nucleus of individual cells can lead to a superior successful rate of expression at the single-cell level in comparison to other nonviral gene delivery methods. Due to the high spatial resolution, the functionalized nanoneedle can also be used to deliver exogenous genes into small cellular compartments, such as axonal terminals or dendritic spines, which have their own gene expression systems independent from those in the neuron cell body (soma).

Small signaling molecules for signaling amplification and transduction: one of the advantages of the functionalized nanoneedle-based delivery is that it can deliver a small number of molecules into cells. The signaling molecules have the capability to amplify the signaling cascades in mammalian cells; however, the quantification of signaling amplification is far from being completely understood. While various theoretical methods has been applied to understand quantification of signaling amplification, most experimental approaches are rudimentary, and yield little information on quantification of any specific cellular events in native environment. Using the functionalized nanoneedle-based delivery, one can deliver small diffusible signaling molecules such as second messenger cyclic AMP, cyclic GMP, inositol phosphates, and diacylglycerol, and monitor how individual second messengers can trigger signaling amplification in a specific cellular setting.

Delivery of polypeptides or proteins for examining protein transportation, stability and degradation: the functionalized nanoneedle-based delivery can also be used to directly deliver polypeptides and proteins into specific cellular compartments. Although the direct delivery of polypeptides and proteins has not been demonstrated with this nanoneedle-based delivery system, it should be attainable by modifying the surface functionalization of the nanoneedle for the purpose of attaching protein molecules [27, 30]. Alternatively, one can exploit the physical and chemical properties in a specific targeting cellular environment to allow the proteins to be absorbed on the needle, and desorbed in the cellular environment. The challenge is to develop different functionalized needle surface for the conjugation of diversified polypeptides or proteins onto the nanoneedle. An additional challenge would be to conjugate large proteins on the nanoneedle for delivery due to the difficulties of handling large proteins in vitro.

Upon successful delivery of a few protein molecules with fluorescent markers into a cell, the movement of the delivered proteins can be potentially tracked to understand the protein distribution and functions at different cellular processes such as cell cycle, cell growth, and protein processes and delivery. In addition, the function of the delivered protein can be monitored with live cell imaging by detection of the substrate production or changes in biophysical and chemical properties, such as ion concentrations. Moreover, the stability of the delivered proteins can be tracked inside of cells. This is particular useful to check protein stability of signaling molecules which can undergo rapid degradation under stimulation of hormones and growth factors.

7. Perspectives

Bio-functionalized nanoneedle-based intracellular delivery presents a novel method to deliver biomolecules into cells with high spatio-temporal resolution. The delivery system can deliver biomolecules to virtually any type of cells with minimal intrusiveness, which makes it feasible to deliver biomolecules into low copy cells such as certain sensory neurons isolated from animals, and thus expands the application of cellular studies on these cells. The method also possesses potential advantages in delivering a few molecules with high spatial and temporal precision, which can enable the single-molecule study of a wide range of cellular processes, including cell cycle, cell growth and differentiation, membrane potential and redox environment, signaling transduction and amplification, and gene expression. It will be critical for the further development of this method for the delivery of a broad range of biomolecules from nucleic acids, proteins, to small diffusible molecules. Meanwhile, it is equally important to integrate the nanoneedle-based delivery system with other experimental techniques such as live cell imaging and patch clamping to broaden the biological application of such high-end techniques to tackle biologically important but challenging problems. In the light of its high transfer efficiency and flexibility in the kind of molecules that can be transferred, it would be interesting to explore the possibilities of automation for this nanoneedle-based delivery, which would allow high-throughput screening required for systematic large scale genome projects.

Beyond intracellular delivery, the nanoneedle can also be used in many other ways for biological experiments in living cells [31]. For example, one may use a conductive nanoneedle, electrically insulated throughout its sidewall but exposed at the very end of its tip, as a nanoscale electrochemical probe to measure electrochemical reactions and signaling processes occurring inside cells or between neighboring cells [32, 81, 82]. As the cellular probes based on glass micropipettes (e.g., used for patch-clamp and microelectrode techniques) have revolutionized cellular electrophysiology, the nanoneedle nanoprobe capable of spatially-resolved electrochemical measurements would further enhance our understanding of related cellular processes.

Acknowledgements

The work was supported by NSF (Grant DMI 0328162, CBET 0933223, and CBET 0731096), the Grainger Foundation, and NIH (Grant GM072744 and HL082646)

Footnotes

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