**4. Conclusions**

Blue, green and red fluorescent nanoparticles composed of thermosensitive liposomes (TSLs) of DPPG and the conjugated polyelectrolytes HTMA-PFP, HTMA-PFBT and HTMA-PFNT respectively, have been prepared and characterized in order to obtain fluorescent drug carriers. In addition, the ability of the nanoparticles for bioimaging applications, transport and control drug delivery has been explored, evidencing their potential use as multifunctional nanoplatforms with imaging and therapeutic functionalities.

The nanoparticles exhibited stable fluorescence signals, good colloidal stability, spherical morphology and hydrodynamic diameters slightly higher to those of DPPG-TSLs, suggesting a membrane surface location of polyelectrolytes, which was confirmed by quenching experiments. In addition, their thermosensitive properties (cooperativity and transition temperature close to 42 ◦C) were similar to those of the DPPG-TSLs, supporting the potential use of these nanoparticles to carry and release drugs triggered by mild hyperthermia. The use of the dye carboxyfluorescein (CF) as a model hydrophilic drug allowed confirmation of this assumption. The dye was entrapped in the aqueous cavity of the fluorescent nanoparticles and was mostly released when nanoparticles were incubated above 40 ◦C. However, a small fraction of dye was slowly released at temperatures near 37 ◦C. This behavior has been attributed to a deeper penetration of the polyelectrolytes in the lipid bilayer, occurring above the pre-transition temperature, which could slightly modify the membrane permeability, allowing the slow release of the dye.

Finally, preliminary experiments with mammalian cells showed the capability of the nanoparticles to mark and visualize cells in blue, green and red colors, extending their applications as bioimaging probes. In this respect, it would be possible to encapsulate a di fferent hydrophilic drug in each type of nanoparticle. This result could be of grea<sup>t</sup> interest in two-photon excitation microscopy and for dynamic imaging of living cells, due to the possibility of simultaneously exciting the fluorescence emission of the three nanoparticles at a single wavelength. We plan to further expand the multifunctionality of the nanoparticles in the future by linking di fferent tumor-targeting molecules, such as peptides, folic acid, antibodies or other small molecules to the fluorescent lipid vesicles. By this procedure, the nanoparticles can be more e ffectively targeted in order to perform di fferential cell marking and active delivery to di fferent tumor sites, which could be visualized in real time.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2079-4991/9/10/1485/s1, Figure S1. Anisotropy values, <*r*>, of DPH in DPPG-TSLs as function of temperature (20–70 ◦C) in sodium phosphate bu ffer, Figure S2. Changes in fluorescence intensity (Δ*I*) of (a) HTMA-PFP (3 μM), (b) HTMA-PFBT (3 μM) and (c) HTMA-PFNT (3 μM) at increasing concentrations of DPPG, Figure S3. Stability kinetics of (a) blue, (b) green and (c) red fluorescent nanoparticles (squares) compared with the stability of the corresponding polyelectrolytes in sodium phosphate buffer (circles), measured at 25 ◦C by monitoring their fluorescence intensity (blue: λexc = 380 nm, λem = 412 nm; green: λexc = 425 nm, λem = 500 nm; red: λexc = 510 nm, λem = 622 nm), Figure S4. Fluorescence emission spectrum of a sample containing simultaneously blue, green and red nanoparticles, upon excitation at 335 nm.

**Author Contributions:** Conceptualization, C.R.M. and M.J.M.-T.; methodology, C.R.M. and M.J.M.-T.; validation, M.R.-C. and Y.A.; formal analysis, C.R.M., M.J.M.-T., M.R.-C. and Y.A.; investigation, M.R.-C. and Y.A.; resources, R.M.; data curation, M.J.M.-T., M.R.-C. and Y.A.; writing—original draft, C.R.M. andM.J.M.-T. with the collaboration of M.R.-C.; writing—review and editing, C.R.M. and M.J.M.-T. with the collaboration of M.R.-C.; visualization, M.R.-C., Y.A. and M.J.M.-T.; supervision, C.R.M. and M.J.M.-T.; project administration, C.R.M. and R.M.; funding acquisition, C.R.M. and R.M.

**Funding:** This research was funded by the Ministerio de Economía, Industria y Competitividad, Gobierno de España (MAT-2017-86805-R), Conselleria d'Educació, Investigació, Cultura i Esport (ACIF/2018/226) and the European Regional Development Fund (IDIFEDER2018/20).

**Acknowledgments:** The authors gratefully acknowledge Alberto Falcó (IDiBE, Elche, Spain) for the kindly donation of the human embryonic kidney cell line HEK293, and Elisa Perez (IDiBE, Elche, Spain) for her kind help and technical assistance.

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