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This review focuses on the recent developments in nanoplasmonic technologies for on-demand and systematic intracellular gene regulation. Types of nanoplasmonic carriers and DNA/RNA cargo are described. Strategies to liberate cargo from their carriers using light are reviewed. Nanoplasmonic technologies enable on-demand and systematic silencing of endogenous intracellular genes. In addition to inhibitory effects, exogenous foreign genes are also introduced and expressed on-demand using nanoplasmonic technologies. The magnitude and timing of genetic activities can therefore be systematically controlled on-demand. Equipped with new nanoplasmonic technologies to directly probe the intracellular space, quantitative approaches should capture many dynamic activities within living systems that were otherwise previously impossible to control using conventional methods.
A single living cell is an amazingly dynamic system that is capable of constantly sensing and responding to its perpetually changing environment. As such, it is an integrated system consisting of extracellular input signals streaming in from the local external environment, interlinked signaling cascades of internal connections, and gene transcriptional networks that respond by producing the appropriate proteins to give rise to cell function (Fig. 1). Quantitatively understanding the inner workings of these systems is the quest set forth by quantitative cell biology and systems biology. By systematically perturbing with extracellular stimuli, specific internal connections can be mapped and the resulting cell function can be observed in response to the single environmental change. Spatial, temporal, and systematic variation of extracellular stimuli has been shown to be absolutely critical for developmental processes , growth , differentiation , apoptosis, and stem cell fate decisions .
In addition to extracellular control of environmental stimuli, intracellular control of the actual internal connections themselves can provide unparalleled insight into the inner workings of these systems. When a cell is treated as input-output “black box,” ideally, the output response is directly correlated to the input signal. However, signal distortions - time delays, noise, and signal magnitude reductions - can confound this input-output relationship. Time delays, due to inherent time-dependent interactions, transcriptional rates, and translational rates, are associated with each interconnected step inside the “black box.” Noise, from stochastic fluctuations in proteins, is introduced at each stage inside the “black box.” Signal magnitude reductions, due to degradation, dilution and diffusion effects, are also inevitable. These signal distortions can have significant implications on the output response. Consider a simple transcriptional network, where gene X regulates gene Y, and gene Y in turn regulates the output. In the presence of its input signal, X becomes active. Threshold effects are known to govern many gene regulatory processes . Thus, when the concentration of X is greater than the threshold concentration required for activating Y, then Y is produced. Subsequently, when the concentration of Y is greater than the threshold concentration required for activating the output, then the output is produced. Now, suppose that the input signal is externally introduced (Fig. 1a). As this extracellular input signal traverses through each step of the extracellular-to-intracellular cascade, signal distortions are introduced. If the signal is significantly confounded by noise or if the signal magnitude is significantly reduced such that the activity of Y no longer satisfies the threshold condition (Fig. 1b), the output is not produced (Fig. 1c). This result can give the misleading impression that no relationship exists between the input signal and Y, when in fact they are related. Internal connections can be incorrectly mapped. Suppose now that Y is directly controlled at the intracellular level (Fig. 1d). Steps in the extracellular-to-intracellular cascade are essentially bypassed, thereby minimizing signal distortions. As a result, the output is reliably produced (Fig. 1e). Internal connections can therefore be correctly mapped and modeled.
By delving directly into the intracellular “black box,” the magnitude and timing of intracellular processes can be precisely controlled. Interfering oligonucleotides, such as antisense DNA, single-stranded RNA, short hairpin RNA (shRNA), and small interfering RNA (siRNA), enable direct, sequence-specific control of intracellular genes, but alone, lack the temporal control necessary for precise manipulation. Recent advancements in chemical biology, nanotechnology, and plasmonics now enable new light-sensitive tools of sub-nanometer and nanometer size scales to directly interface with intracellular processes. For example, nanoplasmonic technologies can be used as carriers of oligonucleotide cargo (Fig. 1d). Initially, oligonucleotide functionality is inactivated. Using light illumination as a remote trigger to release free oligonucleotides and “activate” their functionality, endogenous intracellular genes can be silenced on-demand. In addition to the inhibitory effects of interfering oligonucleotides, exogenous foreign genes can be also introduced and expressed on-demand. In this way, the magnitude and timing of genetic activities can be systematically varied on-demand. Signal distortions can be minimized since light-sensitive tools are activated from within the intracellular space, and therefore, steps in the extracellular-to-intracellular cascade are bypassed at the time of activation. Equipped with new tools to directly probe the intracellular space, quantitative and systematic approaches should capture many dynamic activities within the living cell that were otherwise previously impossible to detect using conventional methods.
Caging is also an effective means to temporarily inactivate oligonucleotide functionality by incorporating photo-labile protective groups, otherwise known as caging groups, into the bases or the phosphate backbone . Ultra-violet (UV) irradiation removes the caging groups and restores oligonucleotide functionality. UV-activated gene silencing using caged antisense DNA has been demonstrated in mouse NIH 3T3 fibroblast cells . UV-activated gene silencing using caged siRNA has also been demonstrated in human HeLa cervical carcinoma cells . While caging enables excellent spatiotemporal control of intracellular genes, the particular use of UV wavelengths is of some concern since intracellular proteins and nucleic acids are widely known to absorb, crosslink, and mutagenize in the presence of UV irradiation. Therefore, activation using less biologically harmful wavelengths of light is highly desirable.
Gold nanoplasmonic particles (GNPs), in the near-infrared (NIR) spectral region, are attractive candidates for intracellular control. The NIR wavelength regime is well suited for biological and biomedical applications since tissues and cells are essentially transparent between 700–1300 nm . Due to their strong and sharp resonance peak in their optical properties, GNPs efficiently convert light energy into surface-localized heat, otherwise known as photothermal conversion [10–12], when the incident light is matched to their plasmon resonance wavelength. In the presence of this incident light, the conduction band electrons of the GNPs collectively oscillate in phase on resonance and subsequently make collisions with the metal lattice, thereby dissipating heat . Heat transfer from the surface of GNPs’ to the surrounding cellular environment is highly localized, decaying exponentially within a few nanometers [10,13,14] and therefore is thought to have minimal adverse effects on cells. Among the GNPs, gold nanorods [13,15,16] and gold nanoshells [17,18], are of widespread interest and pervasive use due to their large absorption cross-section, facile tunability of their plasmon resonance wavelength based on geometry, and large-scale synthesis with uniform distribution (Fig. 2).
Because of their large surface-to-volume ratio, GNPs are ideal carriers of cargo, such as interfering oligonucleotides. While attached to their carriers, cargo is rendered inactive due to steric hindrances between the tightly-packed cargo. For 150 nm diameter gold nanoshells, for example, the surface coverage of dsDNA cargo was determined to be 6400 dsDNA molecules/nanoshell . In the presence of light that is matched to their plasmon resonance wavelength, GNPs photothermally release their cargo to freely interact with the local environment. Several strategies, employing different carrier and cargo types, have been demonstrated (Table 1). For example, it has been shown that short single-stranded DNA, otherwise known as antisense DNA, can be hybridized to a thiolated complementary sense strand, bound to a gold nanorod’s surface through the gold-thiol covalent bond, and photothermally dehybridized using continuous-wave incident light that is matched to the plasmon resonance wavelength of the gold nanorods  (Fig. 3a). Antisense DNA has also been photothermally dehybridized from gold nanoshells  (Fig. 3b) and gold nanoprisms  (Fig. 3c) using continuous-wave illumination. This strategy of photothermal dehybridization using continuous-wave illumination offers several notable advantages. Firstly, no chemical modifications are made to the antisense DNA strand itself since a thiolated complementary strand is used to directly conjugate to the GNP’s surface. Because chemical modifications can interfere with nucleic acid functionality and gene silencing efficacy, unmodified antisense DNA is highly desirable. Secondly, gold-thiol covalent bonds are stable after illumination, such that the GNP’s surface remains covered with the thiolated complementary sense strands. With respect to cytotoxicity, this surface coating of complementary strands after illumination is critical. While the gold core is widely accepted as being biocompatible, bare GNPs have been shown to interact with proteins and induce mis-folding at physiological conditions . Maintaining surface coverage with complementary strands after illumination also prevents reattachment of antisense DNA strands back onto the GNP carrier since rehybridization events are thermodynamically unfavorable due to steric hindrances and electrostatic repulsive forces at the GNP’s surface . Finally, the structural integrity of GNPs is uncompromised after illumination. Maintaining structure after illumination is crucial for in vivo applications, where the size, geometry, coating material, and core material of nanoparticles are precisely designed and carefully characterized for proper biodistribution . Maintaining structure after illumination also allows unique nano-scale optical properties to be retained, thereby enabling the same incident light wavelength to be used. Repetitive or finely-graded release of cargo is conceivable for future applications requiring precise temporal patterns of cargo release. For example, the delivery of a drug at its most effective concentration profile is an on-going challenge. When a drug is introduced, its concentration steadily decreases, producing a large concentration swing between the intervals at which the drug is administered. Photothermal cargo release from GNPs can be used to temporally control drug release for delivering programmable drug concentration profiles that are tailored to specific patients and applications. In addition to temporal control, spatial control of photothermal cargo release from GNPs can also be utilized for localized gene therapy at the tumor location. Genetic abnormalities at the tumor location can be corrected without adversely affecting neighboring, functional organs.
Alternatively, thiol-modified antisense DNA can be covalently bound to gold nanorods directly. It has been demonstrated that pulsed incident light can photothermally melt gold nanorods into spheres, thereby destabilizing the gold-thiol covalent bond and releasing thiol-modified antisense DNA  (Fig. 4a). Photothermal melting has also been effective at releasing thiol-modified linearized plasmid DNA from gold nanorods  (Fig. 4b) and thiol-modified siRNA from gold hollow nanoshells [25,26] (Fig. 4d). Additionally, photothermal melting has been shown to destabilize electrostatically-attached circular plasmid DNA from gold nanorods [27,28] (Fig. 4c). This strategy of photothermal melting using pulsed illumination presents several unique functionalities. Firstly, photothermal melting ensures complete release of all cargo. Secondly, the resulting shape change from rods to spheres enables a distinct and defined shift in the plasmon resonance wavelength. A second light source can be easily employed to re-match to the plasmon resonance wavelength of these shape-transformed GNPs. These functionalities may be useful for future studies that involve multifunctional release and detection schemes. Conceivably, cargo can be completely release from gold nanorods using pulsed illumination, and after a shape transformation, binding activity can then be detected using the now bare gold spherical surface. In the case of gold nanoshells, their silica cores deform after pulsed illumination due to high temperature heating (lattice temperatures reaching ~1064°C, the melting point of bulk gold) . As a result, their plasmon resonance wavelength can also shift after illumination, but in a less distinct and controlled manner compared with photothermally melted gold nanorods. Re-matching the incident light wavelength can therefore be considerably more difficult if the plasmon resonance wavelength cannot be easily controlled after shape transformation.
GNPs enable “nanoplasmonic control” of genetic activities with sequence-specificity and spatiotemporal resolution. GNPs carrying genetic cargo are internalized in living cells by endocytosis. While attached to their carriers, the cargo is temporarily “inactive”. The cargo is also protected from degradation by nucleases due to steric hindrances between the tightly-packed cargo at the GNPs’ surface . Illumination matched to the plasmon resonance wavelength of GNPs is used to “activate” cargo by photothermally disrupting encapsulating endosomes [13,31–34] and photothermally releasing free cargo into the cytosol. In this way, exogenous foreign genes can be introduced and expressed on-demand. Endogenous intracellular genes can also be silenced on-demand.
Nanoplasmonic induction of exogenous EGFP expression has been demonstrated in human HeLa cervical carcinoma cells . This mechanism is illustrated in Figure 5a. Thiol-modified, linearized EGFP-N1 plasmid DNA is covalently bound to gold nanorods and internalized in HeLa cells. Pulsed illumination is used to photothermally melt gold nanorods into spheres, thereby destabilizing the thiol-gold bond and releasing the plasmid into the cytosol. The plasmid transports to the nucleus. Exogenous mRNA is then transcribed from the plasmid, exported out the nucleus, and translated into the corresponding EGFP protein. The nanoplasmonic gene induction of EGFP expression is shown in the fluorescent image in Figure 5b . Thus, expression of exogenous foreign genes can be induced on demand using nanoplasmonic control.
In addition to inducing exogenous expression, endogenous intracellular genes can also be silenced on demand. Nanoplasmonic silencing of endogenous ERBB2 expression using antisense DNA has been demonstrated in human BT474 breast carcinoma cells . As illustrated in Figure 6a, the duplex of thiol-modified sense and unmodified antisense DNA is covalently bound to gold nanorods and internalized in BT474 cells. In the presence of continuous-wave illumination, antisense DNA strands dehybridize and release into the cytosol, while the thiol-modified sense strands remain attached to the gold nanorods. The unbound antisense DNA then binds to a portion of the mature mRNA corresponding to ERBB2. Once the mRNA/antisense DNA heteroduplex is formed, it is recognized and degraded by RNase H enzymes in the cytosol, thereby silencing ERBB2. As seen in Figure 6b, nanoplasmonic silencing of ERBB2 expression is qualitatively demonstrated using immunofluorescence imaging and quantitatively shown using flow cytometry . To ensure light illumination itself and photothermally generated heat had no adverse effects on cells, viability studies were conducted using a fluorescence assay . Since RNase H, is ubiquitously present in both the cytosol and the nucleus [35,36], an alternative model  suggests that unbound antisense DNA transports to the nucleus. In the nucleus, the antisense DNA binds to the premature mRNA. Once this heteroduplex is formed, it is recognized and degraded by RNase H enzymes in the nucleus.
Intracellular genes can also be silenced on-demand using siRNA. siRNA also signals the degradation of specifically targeted mRNA; however, degradation is through a different mechanism compared to antisense DNA. Nanoplasmonic silencing of EGFP expression using siRNA has been shown in mouse endothelial C166 cells stably expressing EGFP . In Figure 7a, thiol-modified siRNA targeting EGFP is covalently bound to gold hollow nanoshells and internalized in endothelial cells stably expressing EGFP. Pulsed illumination is used to photothermally melt gold hollow nanoshells, thereby destabilizing the thiol-gold bond and releasing siRNA into the cytosol. The unbound siRNA sequentially triggers cytosolic RNA-inducing silencing complex (RISC) to unwind the duplex, binds to complementary mature mRNA, and silences EGFP expression. Nanoplasmonic silencing of EGFP in cells stably expressing EGFP is shown in the fluorescent images in Figure 7b . Nanoplasmonic silencing of endogenous genes using siRNA has also been demonstrated in vivo . Thiol-modified siRNA targeting NF-κΒ p65 are covalently bound to hollow gold nanoshells, internalized in Hela cells, and transplanted in mice as xenografts. Pulsed illumination is used to photothermally melt gold hollow nanoshells, thereby destabilizing the thiol-gold bond and releasing the siRNA. Nanoplasmonic silencing of NF-κΒ p65 is shown in the fluorescent images in Figure 7c . Since key components of RISC have been discovered in both the nucleus and cytosol , another model  suggests that unbound siRNA transports to the nucleus. In the nucleus, siRNA triggers RISC to unwind the duplex, binds to premature mRNA, and silences gene expression. Other models  also propose that RISC actively transports siRNA from the cytosol to the nucleus for degradation.
In order to create widespread acceptance of nanoplasmonic technologies by the biology community, optimal plasmonic properties, colloidal stability, and internalization efficiencies of nanoplasmonic technologies must be demonstrated under physiological conditions. Multilayering cargo-carriers with cationic lipids have been shown improve to colloidal stability and internalization under physiological conditions . Thus, in addition to the importance of cargo conjugation to carriers, additional protective surface coating materials also deserve critical attention [39,40]. It must also be demonstrated to the biology community that nanoplasmonic technologies are indeed biocompatible and nontoxic. Toxicology, biodistribution, and environmental studies on the long term effects of nanoplasmonic materials are necessary to promote these technologies and move the fields of nanoplasmonics-enabled quantitative biology and on-demand gene therapeutics forward.
Finally, both extracellular and intracellular controls are necessary to systematically perturb living systems. This integration of extracellular and intracellular controls can provide unprecedented insight into the dynamic nature of living systems for gene regulation and cell reprogramming. For example, microfluidic technologies can provide precise spatial and temporal control of the local external environment of a cell while nanoplasmonic technologies systematically perturb the intracellular space. Dynamic activities can be controlled and biological questions can be answered that were otherwise previously impossible using conventional methods.
The authors acknowledge the Siebel Foundation for graduate support S.E.Lee (Siebel Scholarship, Class of 2010). The authors acknowledge the National Institutes of Health (NIH) Nanomedicine Development Center for the Optical Control of Biological Function (PN2 EY018241) for financial support. The authors thank Prof. Han Lim and Prof. Robert Fischer for insightful discussion on systems biology.
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