*Article* **Polyfluorene-Based Multicolor Fluorescent Nanoparticles Activated by Temperature for Bioimaging and Drug Delivery**

#### **Marta Rubio-Camacho, Yolanda Alacid, Ricardo Mallavia, María José Martínez-Tomé \* and C. Reyes Mateo \***

Instituto de Investigación Desarrollo e Innovación en Biotecnología Sanitaria de Elche (IDiBE), Universidad Miguel Hernández de Elche (UMH), 03202 Elche, Alicante, Spain; marta.rubioc@umh.es (M.R.-C.); yoli2395@gmail.com (Y.A.); r.mallavia@umh.es (R.M.)

**\***Correspondence: mj.martinez@umh.es (M.J.M.-T.); rmateo@umh.es (C.R.M.); Tel.: +34-966-652-475 (M.J.M.-T.); +34-966-658-469 (C.R.M.)

Received: 27 September 2019; Accepted: 15 October 2019; Published: 18 October 2019

**Abstract:** Multifunctional nanoparticles have been attracting growing attention in recent years because of their capability to integrate materials with different features in one entity, which leads them to be considered as the next generation of nanomedicine. In this work, we have taken advantage of the interesting properties of conjugated polyelectrolytes to develop multicolor fluorescent nanoparticles with integrating imaging and therapeutic functionalities. With this end, thermosensitive liposomes were coated with three recently synthesized polyfluorenes: copoly-((9,9-bis(6-*N,N,N*-trimethylammonium)hexyl)-2,7-(fluorene)-alt-1,4-(phenylene)) bromide (HTMA-PFP), copoly-((9,9-bis(6-*N,N,N*-trimethylammonium)hexyl)-2,7-(fluorene)-alt-4,7-(2- (phenyl)benzo(d) (1,2,3) triazole)) bromide (HTMA-PFBT) and copoly-((9,9-bis(6-*N,N,N*trimethylammonium)hexyl)-2,7-(fluorene)-alt-1,4-(naphtho(2,3c)-1,2,5-thiadiazole)) bromide (HTMA-PFNT), in order to obtain blue, green and red fluorescent drug carriers, respectively. The stability, size and morphology of the nanoparticles, as well as their thermotropic behavior and photophysical properties, have been characterized by Dynamic Light Scattering (DLS), Zeta Potential, transmission electron microscope (TEM) analysis and fluorescence spectroscopy. In addition, the suitability of the nanostructures to carry and release their contents when triggered by hyperthermia has been explored by using carboxyfluorescein as a hydrophilic drug model. Finally, preliminary experiments with mammalian cells demonstrate the capability of the nanoparticles to mark and visualize cells with different colors, evidencing their potential use for imaging and therapeutic applications.

**Keywords:** multifunctional fluorescent nanoparticles; conjugated polyelectrolytes (CPEs); thermosensitive liposomes (TSLs); bioimaging; drug carrier; release experiments
