**3. Results**

#### *3.1. Preparation of Cationic Nanogels (NANOGEL-1 and NANOGEL-2) and Cationic Fluorescent Nanogel Thermometers (NANOGEL-3~6)*

The cationic nanogels (NANOGEL-**1** and NANOGEL-**2**) and cationic fluorescent nanogel thermometers (NANOGEL-**3**~**6**) were synthesized by radical polymerization, due to the cationic radical initiator ADIP with the starting materials indicated in Table 1. Crude nanogels were purified by reprecipitation with a salting-out technique and subsequent dialysis for at least one week. The purity of NANOGEL-**1**~**6** was confirmed by 1H-NMR measurements (Figure S1 in Supplementary Materials). The moderate yields of NANOGEL-**1**~**6** likely resulted from their substantial loss during the reprecipitation (due to high hydrophilicity of NANOGEL-**1**~**6** with cationic surfaces) and the long-term dialysis. The size and surface charge were evaluated by TEM/DLS measurements and zeta potential measurements (Figure 2), respectively, and are summarized in Table 2. The temperature-dependent hydrodynamic diameters of NANOGEL-**1**~**6** determined by DLS clearly show the thermosensitive characteristics of the polyNIPAM units (cf., volume phase transition temperature of the polyNIPAM gel is 32 ◦C [34]). Sufficient amounts of cationic terminals introduced by ADIP to increase the solubility of nanogels resulted in the isolation of nanogels at a high temperature (45 ◦C) without aggregation (Table 2). NANOGEL-**1** without fluorescent DBD-AA units was obtained by referring to the concentrations of monomers adopted for the preparation of anionic NIPAM nanogels [15]. Interestingly, the cationic NANOGEL-**2** could be obtained without CTAC, which is a rare example in the preparation of NIPAM nanogels without any surfactant molecules [35]. Then, the cationic fluorescent nanogel thermometers NANOGEL-**3**~**6** were synthesized with various concentrations of an environment-sensitive fluorescent monomer, DBD-AA. The amounts of fluorescent DBD-AA units in NANOGEL-**3**~**6** were converted to the corresponding concentrations when the nanogel concentration was 0.01 w/v% (Table 2).

**Figure 2.** Characterization of NANOGEL-**1** as a representative. (**a**) TEM image; (**b**) size distribution measured by DLS (0.001 w/v% in water at 25 and 45 ◦C); (**c**) zeta potential distribution (0.1 w/v% in water at 45 ◦C).

#### *3.2. Fluorescence Responses of Cationic Fluorescent Nanogel Thermometers (NANOGEL-3~6) in Aqueous Solutions*

The fluorescence spectra of NANOGEL-**3**~**6** (0.01 w/v%) in water and 150 mM KCl solution were recorded with changing temperature (Figure 3). The fluorescence intensity ratio at 25 and 45 ◦C (defined as the fluorescence enhancement (FE) factor) and the maximum emission wavelengths at 25 and 45 ◦C are listed in Table 2. All samples showed fluorescence enhancements at approximately 32 ◦C, which is the lower critical solution temperature of PNIPAM nanogels [34]. The heat-induced fluorescence enhancement of NANOGEL-**3**~**6** in 150 mM KCl solution was higher than that in water because of the salting out effects. It is known that salts, including KCl accelerate the dehydration of NIPAM units by hydrogen bonding with their amide groups and by increasing the surface tension of water in the hydration shell around the hydrophobic groups [36,37]. Nevertheless, the salting out effects by KCl

on the fluorescence responses of NANOGEL-**3**~**6** were gradually saturated when the concentration of KCl exceeded 50 mM (Figure S2), which is preferred for the use under intracellular conditions (where [K+] is approximately 139 mM [38].) As indicated in Figure 3a and Table 2, the maximum emission wavelength at a high temperature (i.e., 45 ◦C) was remarkably shorter than that at a low temperature (i.e., 25 ◦C). This temperature-dependent maximum emission wavelength is due to the functional mechanism of NIPAM-based fluorescent polymeric thermometers [6], i.e., the drastic variation of the microenvironment near DBD-AA units with a heat-induced structural change in NIPAM units. As cationic fluorescent nanogel thermometers contain many fluorescent DBD-AA units, the heat-induced fluorescence enhancement became less with a small shift in the maximum emission wavelength between 25 and 45 ◦C (e.g., NANOGEL-**3** vs NANOGEL-**6** in Table 2). The decrease in sensitivity to the temperature variation is likely due to a structural disturbance of the thermoresponsive nanogels by the relatively bulky DBD-AA units.

**Table 2.** Physical and photophysical properties of cationic nanogels (NANOGEL-**1** and NANOGEL-**2**) and cationic fluorescent nanogel thermometers (NANOGEL-**3**~**6**).


1 Determined by DLS. The av ± s.d. of peak values in triplicate measurements. 2 At 45 ◦C. The av ± s.d. of peak values of triplicate measurements. 3 When the nanogel concentration is 0.01 w/v%. 4 Fluorescence enhancement factor calculated as fluorescence intensity at λem at 45 ◦C divided by that at 25 ◦C.

#### *3.3. Introduction of Cationic Fluorescent Nanogel Thermometers into Mammalian HeLa Cells*

The method for introducing cationic fluorescent nanogel thermometers was optimized using the most strongly fluorescent NANOGEL-**6** and HeLa cells as model mammalian cells. One should note that weakly fluorescent nanogel thermometers with fewer DBD-AA units (e.g., NANOGEL-**4**) could not be detected inside the HeLa cells at a low temperature (e.g., 30 ◦C) with the same laser excitation power and detection sensitivity of the confocal laser scanning microscope used for the detection of NANOGEL-**6**. Figure 4 shows the effect of incubation time at 25 ◦C on the incorporation efficiency of NANOGEL-**6** when established standard conditions for cationic fluorescent polymeric thermometers (0.05 w/v% in a 5 % glucose solution [28]) were adopted. A high temperature (e.g., 37 ◦C) accelerated the introduction of cationic fluorescent nanogel thermometers into the HeLa cells, but induced unfavorable aggregation inside the cells. Therefore, we fixed the incubation time and temperature to be 20 min and 25 ◦C, respectively, in the subsequent experiments. Figure 5 displays the representative transmission and confocal fluorescence images of a HeLa cell containing NANOGEL-**6**, in which the mitochondria were additionally stained by MitoTracker DR. The cationic nanogel thermometer NANOGEL-**6** was detected in the form of dots in the HeLa cells and remained inside them once introduced. While a significant background noise was detected when cationic fluorescent nanogel thermometer NANOGEL-**6** was introduced into adherent HeLa cells, due to the attachment of NANOGEL-**6** on the surface of a glass-bottom dish, treatment of suspended HeLa cells with NANOGEL-**6** could significantly reduce this background noise (Figure 6). This alternative protocol, i.e., introducing NANOGEL-**6** into suspended HeLa cells, is considered for improving the sensitivity of intracellular thermometry by increasing the signal-to-noise ratio.

**Figure 3.** Fluorescence response of NANOGEL-**3**~**6** to temperature variation. (**a**) Temperaturedependent fluorescence spectra of NANOGEL-**3** and NANOGEL-**6** in water and 150 mM KCl aqueous solution. The vertical units in (**a**) are identical.; (**b**) Fluorescence intensity of NANOGEL-**3**~**6** in water (open circle) and 150 mM KCl aqueous solution (closed circle) at λem at 45 ◦C (see Table 2). The inset for NANOGEL-**6** is vertically expanded. Concentration of NANOGEL-**3**~**6**: 0.01 w/v%; excitation: 456 nm. The shoulders at approximately 684 nm in the fluorescence spectra at 40 and 45 ◦C in panel (**a**) were due to the scattered excitation light.

**Figure 4.** Effects of the incubation time on the incorporation efficiency. The HeLa cells were treated with NANOGEL-**6** (0.05 w/v%) in 5 % glucose solution at 25 ◦C for the indicated durations. The vertical bars represent the s.d. evaluated by at least four independent experiments.

**Figure 5.** Cationic fluorescent nanogel thermometer NANOGEL-**6** in a live HeLa cell. (**a**) Transmitted light image; (**b**) confocal fluorescence image of NANOGEL-**6** (excitation: 458 nm; emission: 500–600 nm); (**c**) confocal fluorescence image of mitochondria visualized with Mito Tracker DR (excitation: 633 nm; emission: 645–730 nm); (**d**) merged image.

**Figure 6.** Reduction of background noise in the fluorescence image of HeLa cells with NANOGEL-**6** by the modified incorporation method using suspended cells. (**a**) Transmitted light image (left) and confocal fluorescence image (right, excitation: 458 nm; emission: 500–600 nm) at 37 ◦C when NANOGEL-**6** was introduced into adherent HeLa cells attached on a dish.; (**b**) Transmitted light image (left) and confocal fluorescence image (right, excitation: 458 nm; emission: 500–600 nm) at 37 ◦C when NANOGEL-**6** was introduced into suspended HeLa cells before scattering on the dish. The confocal fluorescence images were focused on the glass surface of the dish. Color bars in the fluorescence images indicate the fluorescence intensity (arbitrary unit).

#### *3.4. Functions of Cationic Fluorescent Nanogel Thermometers inside HeLa Cells*

The functions of NANOGEL-**6** in HeLa cells were examined as a representative cationic fluorescent nanogel thermometer. Similar to the response in aqueous solutions, the cationic fluorescent nanogel thermometer NANOGEL-**6** introduced into HeLa cells showed remarkable fluorescence enhancement when the temperature of the culture medium was increased (Figure 7).

Another unique property of cationic fluorescent nanogel thermometers prepared by ADIP is non-cytotoxicity. As demonstrated in Figure 8, the HeLa cells containing NANOGEL-**6** were capable of dividing in a similar manner to those without staining (i.e., control). The fluorescence response of NANOGEL-**6** to temperature variation was confirmed for the HeLa cells incubated for one day after the introduction of NANOGEL-**6** (Figure 8).

**Figure 7.** Fluorescence response of NANOGEL-**6** in live HeLa cells. (**a**) Transmitted light image at 37 ◦C; (**b**) confocal fluorescence image (excitation: 458 nm; emission: 500–600 nm) at 24 ◦C (left), 30 ◦C (middle) and 37 ◦C (right). Color bars in the fluorescence images indicate the fluorescence intensity (arbitrary unit).

**Figure 8.** Non-cytotoxicity and long-term functionality of NANOGEL-**6** in live HeLa cells. (**a**) Growth of HeLa cells containing NANOGEL-**6** and none (control) (mean ± s.d.); (**b**) Transmitted light image at 37 ◦C (left) and confocal fluorescence image (excitation: 458 nm; emission: 500–600 nm) at 24 ◦C (middle) and 37 ◦C (right) at 24 h after the introduction of NANOGEL-**6** into the cells. Color bars in the fluorescence images indicate the fluorescence intensity (arbitrary unit).
