Mapping Photothermally Induced Gene Expression in Living Cells and Tissues by Nanorod-Locked Nucleic Acid Complexes

The photothermal effect of plasmonic nanostructures has numerous applications, such as cancer therapy, photonic gene circuit, large cargo delivery, and nanostructure-enhanced laser tweezers. The photothermal operation can also induce unwanted physical and biochemical effects, which potentially alter the cell behaviors. However, there is a lack of techniques for characterizing the dynamic cell responses near the site of photothermal operation with high spatiotemporal resolution. In this work, we show that the incorporation of locked nucleic acid probes with gold nanorods allows photothermal manipulation and real-time monitoring of gene expression near the area of irradiation in living cells and animal tissues. The multimodal gold nanorod serves as an endocytic delivery reagent to transport the probes into the cells, a fluorescence quencher and a binding competitor to detect intracellular mRNA, and a plasmonic photothermal transducer to induce cell ablation. We demonstrate the ability of the gold nanorod-locked nucleic acid complex for detecting the spatiotemporal gene expression in viable cells and tissues and inducing photothermal ablation of single cells. Using the gold nanorod-locked nucleic acid complex, we systematically characterize the dynamic cellular heat shock responses near the site of photothermal operation. The gold nanorod-locked nucleic acid complex enables mapping of intracellular gene expressions and analyzes the photothermal effects of nanostructures toward various biomedical applications.

Dividing the number of GNRs determined by UV-Vis extinction measurement yield the number of LNA probe attached to a single GNR to approximately 34 LNA per GNR.
Considering each GNR carries ~34 copies of LNA probes in a cell, the final probe concentration inside a cell was approximately 45 nM. For cell imaging, 2×10 11 GNR/ml GNR-LNA complexes targeting heat shock protein 70 (HPS70) mRNA, β-actin mRNA, and random sequence were tested.

Quantification of internalized GNRs
Inductively coupled plasma mass spectrometry (ICP-MS) was applied to quantify GNRs internalizations. Briefly cells were cultured overnight. GNRs with concentration of 10 10 , 2×10 11 , and 5×10 11 GNR/ml were introduced to 24 well plates and incubated for 12 hours. Cells were then washed for 3 times with 1X PBS to remove excessive GNRs that were not internalized. Cells were harvested with 0.25% trypsin solution and centrifuged at 1500 rpm for 5 min. The cell pellets were then resuspended in 1X PBS to obtain a final concentration of 5×10 5 cell/ml. The GNR samples were prepared by acid digestion of cells using the CEM MARS6 microwave digestion system (Matthews, N.C.).
The sample was weighed in a tared Teflon vessel. Freshly-prepared aqua regia (Aristar-Plus grade HNO3:HCl at 1:3 ratio) was added. After one hour of pre-digestion, 1.0 ml of 18 megohm water was added prior to sealing the vessels. During the digestion process, the temperature of the solution reached to 200 o C after 20 min, and was held at this temperature for an additional 15 min. After cooling, the digest was transferred into a pre-weighed 15mL trace-metal-free PP tube (VWR Scientific). The mass of GNRs in the solution were then determined by Agilent 7700x CIP-MS system (Agilent Technologies, Santa Clara, CA) and converted to the number of GNRs per cell using the calculated number of gold atom in each nanorods according to the equation previously reported 10 .

Statistical analysis.
All experiments were performed independently for at least three times. For mapping the spatial and temporal heat shock response of cells after laser irradiation, at least 50 cells were measured for each data point. The data are expressed as mean ± SEM. For characterizing laser induced temperature elevation, the experiments were repeated (n=3) for statistical analysis. Statistical analysis was carried out using Student's t-test.

Temperature measurement
The temperature was measured at different time points and locations from the laser.
For thermal heating and cooling experiments, miniaturized thermocouples (Omega) with 10 µm diameter were placed inside the media near the cell monolayer. Data were recorded using a thermocouple reader (Okolab) at different time points. The thermocouple was placed at different locations using a costumed 3D traversing system to determine the temperature distribution near the laser. To verify the temperature measurement and resolve the spatial temperature distribution, infrared thermometry was also performed using a mid-wavelength infrared camera (SC6700, FLIR Thermovision

B. Numerical analysis of temperature profile Heat equation
To investigate the temperature distribution near the site of photothermal operation and the effects of laser power and duration, we performed computational analyses to estimate the temperature profile near the irradiation area. In particular, the heat diffusion equation, which describes the distribution of temperature over time, was considered in the analysis 11 . A heating term was introduced to represent the localized heat generated due to the photothermal effect (equation S1).
where T is the temperature distribution and S is the local photothermal heating source.
D is the thermal diffusivity of the medium. Thermal diffusivity of water was used in the calculation. The value can be modified to describe the heat transfer in other media.
The localized heating is modeled by assigning an appropriate temperature at the location of photothermal operation. The partial differential equation was written in twodimensional form to approximate the photothermal ablation experiment (equation S2).
The equation was discretized in space and time using the finite difference approach and implemented in MATLAB (equation S3). 12 n, i, and j are discrete time and space indexes.

Boundary and initial conditions
Supplementary Fig. S4a shows the domain considered in the simulation. In the simulation domain, there were 20 by 80 elements and each element was 100 µm by 100 µm in size. Initial and boundary conditions were required to solve the heat equation.
For initial condition, the temperature was considered to be uniform at the reference temperature initially. The thermal boundary condition was determined by the heat transfer through the cover slip ( Supplementary Fig. S4b). In particular, the heat loss to the cover glass was determined by the heat transfer coefficients at the liquid-glass interface and glass-air interface, and conductive heat transfer through the cover glass.
T f , T s , T b , and T ∞ were the temperature of the liquid, the liquid-glass interface, the glassair interface, and the ambient. In our experimental condition, the heat flux was mainly determined by the heat transfer coefficient at the glass-air interface. Since most of the heat loss occurred near the laser source on the cover glass, the heat transfer was considered to be the same in all boundaries. The parameters and values used in the numerical simulation are described in Table S2. 11

Temperature distribution with continuous laser irradiation
The temperature profiles with continuous laser heating were shown in Fig. 2h in the main text (see also Supplementary movie S3). In the simulation, heat diffused rapidly to the surrounding media in the first few seconds. The temperature profile then gradually reached a quasi-steady state value when the heat flux is approximately equal to the heat loss to the substrate. Supplementary Fig. S5 shows the temperature profiles near the laser. The region of temperature elevation was localized near the heat source. The temperature dropped to 50% of the maximum value in 200-300 µm, representing a characteristic heat transfer length scale of the system. This length scale was in good agreement with the value measured in the experiment by infrared thermometry and miniaturized thermocouples (see Fig. 2e).

Heating and cooling time constants
To estimate the thermal time constants

Effects of laser duration
We then investigated the effects of the duration of heating on the temperature profile.

C.
Supplementary Tables   Table S1. Sequences of single-stranded LNA probes with alternating DNA/LNA monomers for intracellular detection. Bold italic letters represent LNA monomers.