**4. Discussion**

In the present study, we demonstrated the synthesis of cationic nanogels (NANOGEL-**1**~**6**) and presented a functional assessment of cationic fluorescent nanogel thermometers (NANOGEL-**3**~**6**). One of the important advantages of fluorescent polymeric thermometers is the capability of functional integration by introducing additional units into a macromolecule. In the case of NANOGEL-**3**~**6**, the NIPAM units were assumed to be sensitive to temperature variations, whereas the DBD-AA units produced a fluorescence signal. The crosslinker MBAM units provided the robustness of nanoparticles. The positive charges on the surface, which originated from the radical initiator ADIP, enabled spontaneous entry into mammalian cells. Non-cytotoxicity was also a consequence of this cationic surface of the nanogel. Targeting to organelles by attaching a specific signal on the surface or signal normalization by introducing a second reference fluorophore [39] will be expected to improve in the future.

Table 3 summarizes both the advantages and disadvantages of the four types of fluorescent polymeric thermometers ever developed. Now, we can fill the last empty column in Table 3. In general, cationic fluorescent thermometers are more useful than anionic ones because the former can be introduced into mammalian cells under mild conditions without a microinjection technique so that a large number of samples can be treated. Fluorescent nanogel thermometers and fluorescent linear polymeric thermometers are complementary: fluorescent nanogel thermometers show low toxicity to live cells, but the spatial resolution in intracellular thermometry is low, while fluorescent linear thermometers show opposite characteristics. Recently, biological and even medical researchers have

begun to utilize fluorescent polymeric thermometers in their own studies [40–42]. The cationic fluorescent nanogel thermometers developed in this study will contribute to the field of intracellular thermometry, due to their remarkable non-cytotoxicity, which will enable long-term observations.


**Table 3.** Comparison of fluorescent polymeric thermometers for intracellular thermometry.

1 Neutral fluorescent polymeric thermometers cannot be utilized for intracellular thermometry, due to serious aggregation under the high ionic strength inside living cells. 2 The average temperature of a single cell can be monitored. 3 The temperature distribution inside a cell can be imaged. 4 Interfering with cell division.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2073-4360/11/8/1305/s1, Figure S1: 1H-NMR charts of NANOGEL-**1**~**6**, Figure S2: Effects of the KCl concentration on fluorescence responses of NANOGEL-3 as a representative. The fluorescence intensity is normalized at 25 ◦C. The samples were excited at 456 nm.

**Author Contributions:** Conceptualization, S.U.; methodology, S.U.; validation, T.H., K.K. and S.U.; formal analysis, T.H. and S.U.; investigation, T.H., K.K., N.I. and S.U.; resources, N.I. and S.U.; writing—original draft preparation, S.U.; writing—review and editing, T.H., N.I. and S.U.; visualization, T.H. and S.U.; project administration, N.I. and S.U.; funding acquisition, N.I. and S.U.

**Funding:** This research was funded by the JST (Development of Advanced Measurement and Analysis System) and JSPS (Grant-in-Aid for Scientific Research (B) (17H03075)).

**Conflicts of Interest:** The authors declare that they have no conflict of interest.
