**1. Introduction**

Nanomedicine is an emergen<sup>t</sup> area which results from the application of nanotechnology to medicine. Research in this field has experienced rapid growth during the last decade, extending its applications in bioimaging, disease treatment and diagnosis. A number of nanoformulations for diagnostics and therapeutics have been approved for use in humans, and even more are currently under investigation [1–3]. An important trend in this field is the development of multifunctional nanoplatforms integrating different properties, such as imaging and therapeutic functionalities, in one entity [4,5]. In this regard, a large variety of biocompatible materials including lipids, proteins, carbon and quantum dots, synthetic polymers, fluorophores, dendrimers or metallic nanoparticles have been coupled and organized in nanostructures forming vesicles, micelles, nanorods, dendrimers and more, to be used as nanomedical vehicles in theragnostic applications [6–8].

The main component of these nanostructures is the carrier, which is responsible for transporting the drug and releasing it. Since their discovery in the mid-1960's, liposomes, which are lipid vesicles formed by one or more concentric lipid bilayers surrounding an aqueous core, have been considered to be the most successful nanocarriers for drug delivery, especially in anticancer chemotherapy [9,10]. This success is mainly due to the numerous advantages they o ffer. On one hand, liposomes are highly versatile, allowing the incorporation of hydrophobic drugs in the lipid bilayer or hydrophilic drugs in the aqueous core, as well as surface modifications in order to control their interactions with biological targets. On the other hand, the lipid membrane can behave as a barrier contributing to protect the encapsulated drug from degradation, thus prolonging its half-life in the bloodstream. In addition, due to the enhanced permeability and retention (EPR) phenomenon displayed by tumor tissues in comparison with normal tissues, liposomes can selectively accumulate in the tumor, enhancing e fficacy and minimizing adverse side e ffects [11–13].

The release of drugs from the liposomal formulation is usually very low; nevertheless, the fact that the physical properties of the lipid membrane respond to a wide range of internal and external stimuli (temperature, pH, light, pressure, ions, magnetic field, etc.) can be used to selectively release the drug in a controlled way [14]. One well-established approach for triggering the encapsulated drug release, benefits from the di fferences in the permeability of the lipid membrane between the gel and fluid phase. In the gel phase, lipids are closely packed, with the acyl chains extended, and there is little lateral di ffusion and low permeability. On the contrary, in the fluid phase, the acyl chains are more kinked, packing is lost, and the permeability is relatively high. The gel–fluid phase transition occurs cooperatively at the transition temperature (T m), so it is possible to increase the permeability of the lipid bilayer by increasing the temperature beyond T m, allowing the release of entrapped drugs [15,16]. The permeability is additionally increased just at this temperature, as a consequence of the coexistence of membrane areas in both phases [17].

Liposomes which release their contents at specific temperatures are called thermosensitive liposomes (TSLs) [18,19]. Among the di fferent TSLs reported in the literature, the most clinically preferable are those having their T m between 39–43 ◦C (mild hyperthermia), because these temperatures improve drug uptake, increase tumor perfusion and rend cancer cells temporarily sensitive to other treatments [20,21]. Typical TSLs have been mostly prepared from 1,2-dipalmitoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DPPG) and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) derivatives, alone or combined with other lipids or polymers, due to its drug encapsulation capacity, excellent biodegradability and because their T m occurs at 41–42 ◦C [22,23]. In the hyperthermia treatment, the tumor is locally heated to 40–43 ◦C for a defined period of time. The drug circulating in the bloodstream is safely entrapped in the TSLs because the liposomal membrane is in the gel phase, but once it reaches the heated tumor, the phase transition takes place and the drug is released, generally by passive transfer across the membrane according to a concentration gradient [24].

Incorporation of reporter groups—such as fluorescent components—in drug delivery carrier systems is of grea<sup>t</sup> interest in the fabrication of multifunctional nanoplatforms since they can act as probes for bioimaging and labelling (diagnosis) while monitoring the pathway concerning the drug, providing information from its final location [25,26]. During the last decades, these fluorescent components have been usually small organic fluorophores, fluorescent proteins and inorganic quantum dots (QDs) [27]. In contrast to organic dyes and fluorescent proteins, QDs do not su ffer from photobleaching, self-quenching or chemical degradation and show unique properties such as broad absorption bands, high molar extinction coe fficients, narrow emission peaks and large Stoke shifts. In addition, QDs made up of the same material have distinct emission wavelengths, depending on their size, shifting to the red as the size of QDs increases. These properties allow to simultaneously excite mixed QDs populations with di fferent emission wavelengths at a single wavelength, facilitating multicolor fluorescence imaging [28]. Nonetheless, the potential cytotoxicity risk associated with the chemical composition of QDs (cadmium, selenium, tellurium, etc.) remains the major limitation for using these fluorescent nanoparticles in biological approaches [29–31]. To overcome this concern, it is necessary to develop safe and more e fficient fluorescent carriers which, in addition to encapsulating the drug, incorporate new fluorescent materials with improved characteristics, such as nontoxicity, stability, high sensitivity, etc.

In comparison with common organic dyes, fluorescent proteins and QDs, conjugated polyelectrolytes (CPEs) have unique physiochemical properties. These materials are polymers with highly electron-delocalized backbones, containing ionic side groups which facilitate their water solubilization. CPEs have the optoelectronic properties from their neutral counterpart conjugated polymers (CPs), showing high-fluorescent quantum yields, broad absorption and emission spectra, large Stokes shift, good photostability, and more e fficient intramolecular/intermolecular energy transfer than common organic dyes and fluorescent proteins, while having the advantage of being more biocompatible than QDs [32]. Contrary to CPs, CPEs display the typical physicochemical behavior of polyelectrolytes in aqueous solvents, allowing the coupling with di fferent biological systems via electrostatic interactions [33]. In addition, CPEs have easily tunable side chains for bio-conjugation with several recognition elements and show high versatility in their synthesis, enabling fine-tuning of their absorption and emission bands through backbone modification [34]. Given these properties, CPEs have been successfully applied for detection of a wide range of biological and chemical molecules, but also as novel fluorescent probes for bioimaging, and exhibit enormous potential for therapeutic applications and/or diagnostics [35–38].

Fluorene-based conjugated polyelectrolytes, being fluorescent, nontoxic and photostable, provide excellent thermal and chemical stability. Furthermore, fluorene-based CPEs have good synthetic accessibility at the C9 position of the fluorene ring. Polyfluorenes usually emit in the blue spectral region, but copolymerization with other aromatic units allows for shifting the emission spectrum to longer wavelengths [39–41]. On this matter, we have synthesized three cationic polyfluorenes which emit in the blue, green and red regions of the visible spectrum: copoly-((9,9-bis(6 -*N,N,N*-trimethylammonium)hexyl)-2,7-(fluorene)-alt-1,4-(phenylene)) bromide (HTMA-PFP), which incorporate a phenyl group on fluorene backbone, 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), which incorporates the chromophore 2-phenylbenzotriazole on the backbone 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), which incorporates a naphtha(2,3c) (1,2,5)thiadiazole group on fluorene backbone [42–44] (Scheme 1, upper part). The three CPEs have been extensively characterized, showing interesting properties as fluorescent membrane markers for bioimaging studies [42–46]. In addition, they have common absorption bands around 330–350 nm (Scheme 1, bottom part), therefore, they could be excited simultaneously upon UV excitation, allowing multicolor fluorescence imaging. Finally, because of their cationic charge, the synthesized CPEs have high a ffinity to anionic species, forming complexes with certain proteins and lipid vesicles [33,43].

**Scheme 1.** Chemical structures (**upper par<sup>t</sup>**) and normalized absorption spectra (**bottom par<sup>t</sup>**) of conjugated polyelectrolytes (CPEs) copoly-((9,9-bis(6-*N,N,N*-trimethylammonium)hexyl)-2,7- (fluorene)-alt-1,4-(phenylene)) bromide (HTMA-PFP) (in blue), 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) (in green) 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 red).

In this work, we have taken advantage of the interesting properties of the fluorescent CPEs to develop a multifunctional nanoplatform able to integrate imaging and therapeutic functionalities in one entity. With this end, TSLs composed of the anionic lipid DPPG have been prepared and coated with HTMA-PFP, HTMA-PFBT and HTMA-PFNT, in order to obtain blue, green and red fluorescent drug carriers, respectively. The stability, size and morphology of the nanoparticles have been characterized, as well as their photophysical properties and thermotropic behavior. In addition, the suitability of the nanoparticles as carrier systems to release a drug in response to external mild hyperthermia has been explored using carboxyfluorescein (CF) as a model hydrophilic drug. Finally, preliminary experiments have been carried out to evaluate the capacity of the nanostructures to mark and visualize mammalian cells in different colors.

#### **2. Materials and Methods**

## *2.1. Materials*

The synthetic phospholipid 1,2-dipalmitoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DPPG) was purchased from Sigma-Aldrich (St. Louis, MO, USA). The polyfluorenes HTMA-PFP (Mn (g·mol−1) = 4170; Mw (g·mol−1) = 8340), HTMA-PFBT (Mn (g·mol−1) = 4584; Mw (g·mol−1) = 8531) and HTMA-PFNT (Mn (g·mol−1) = 4507; Mw (g·mol−1) = 8990) were synthesized and subsequently characterized in our laboratory [43,44,47]. Stock solutions of the polyfluorenes were dissolved in dimethyl sulfoxide (DMSO) with a final concentration of 3.65 × 10−<sup>4</sup> M for HTMA-PFP and HTMA-PFNT, and 6.24 × 10−<sup>4</sup> M for HTMA-PFBT (in repeat units), and stored at −20 ◦C before

use. The dye 5(6)-carboxyfluorescein (CF) was purchased from Sigma-Aldrich (St. Louis, MO, USA), as well as the fluorescent membrane probe 1,6-diphenyl-1,3,5-hexatriene (DPH), and the quencher 9,10-anthraquinone-2,6-disulfonic acid (AQS). Stock solutions of the three compounds were prepared in dimethyl sulfoxide (DMSO) (1.25 M), dimethylformamide (DFM) (1 mM) and water (5 mM), respectively. Phosphate bu ffer solution (50 mM, 0.1 M NaCl, pH 7.4) was prepared in Milli-Q water. The rest of the chemicals were of spectroscopic or analytical reagen<sup>t</sup> grade.
