*2.2. Methods*

#### 2.2.1. Preparation of Thermosensitive Liposomes (TSLs)

Lipids solutions of 2 mg of DPPG were left to dry at room temperature. Straight away, the dried phospholipid was resuspended in sodium phosphate bu ffer to a final concentration of 0.5 mM in order to obtain multilamellar vesicles (MLVs). MLVs were then heated above the phospholipid phase transition (~41 ◦C) and vortexed several times. TSLs were obtained from MLVs by pressure extrusion through 0.1 μm polycarbonate filters above T m (~41 ◦C) in order to obtain homogenous large unilamellar vesicles (LUVs).

#### 2.2.2. Preparation of Multicolor Fluorescent Nanoparticles

Aliquots of CPEs solubilized in DMSO (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) were externally added to the TSLs suspension and incubated for at least 30 min at room temperature, as is depicted in Scheme 2. The proportion of DMSO in final samples was lower than 1% (*v*/*v*) in all the cases, and the final CPEs concentration was 3 μM in terms of repeat units.

**Scheme 2.** Preparation of multicolor fluorescent nanoparticles.

#### 2.2.3. Drug Encapsulation and Release Assays

CF was used as a hydrophilic model drug to investigate the encapsulation and controlled release properties of the TSLs and multicolor fluorescent nanoparticles. Encapsulation of the compound was carried out as is depicted in Scheme 2. Briefly, TSLs composed of DPPG were prepared with CF encapsulated in the aqueous core at a concentration of 40 mM in sodium phosphate bu ffer. The non-encapsulated CF was taken o ff by using a gel filtration column loaded with Sephadex G-75 and eluted with sodium phosphate bu ffer. Aliquots of CPEs were externally added to the TSLs loaded with CF, as was previously described. Self-quenching is expected when the CF is entrapped inside the vesicles. The CF release was assayed by treating the CF-loaded vesicles with increasing temperatures up to 60 ◦C. Samples were excited at 492 nm in order to minimize the absorbance of CPEs, and the

emission was collected between 500–550 nm. The amount of CF released during and after thermal treatment was determined by inducing the total breakdown of the vesicles with Triton X-100 at 10%.

#### 2.2.4. Particle Size and Zeta Potential

The size and Zeta Potential of the TSLs and multicolor fluorescent nanoparticles was explored by Dynamic Light Scattering (DLS) technique, with a Malvern Zetasizer Nano-ZS instrument (Worcestershire, UK) equipped with a monochromatic coherent 4 mW Helium Neon laser (λ = 633 nm) light source, where size measurements were performed at angles of 173◦. Size was measured in disposable cuvettes, while Zeta Potential measurements were performed in specific Zeta Potential cells. All measurements were carried out in triplicate at room temperature.

#### 2.2.5. Fluorescence Experiments

Fluorescence measurements were carried out in a PTI-QuantaMaster Spectrofluorometer (Birmingham, AL, USA) equipped with a Peltier cell holder. 1 cm path length quartz cuvettes were used to place the samples, which were subsequently excited at 380 nm (HTMA-PFP), 510 nm (HTMA-PFNT), 425 nm (HTMA-PFBT) or 492 nm (CF). Background intensities were checked and removed from the samples when it was necessary.

#### 2.2.6. Anisotropy Experiments

Changes in the anisotropy as a function of temperature were used to explore the phase transition cooperativity and T m of TSLs. Steady state anisotropy, <*r*>, is defined as:

$$ = \frac{(I\_{VV} - GI\_{VH})}{(I\_{VV} + 2GI\_{VH})} \tag{1}$$

where, *IVV* and *IVH* correspond to the fluorescence intensities collected with the excitation polarizer oriented in a vertical position, and the emission polarizer oriented in a vertical and horizontal position, respectively. These measurements were obtained using Glan–Thompson polarizers incorporated in the spectrofluorometer. The liposome samples in the presence of DPH were excited at 360 nm and the emission was collected at 430 nm. The G factor ( *G* = *IHV*/*IHH*) corrects the transmissivity bias introduced by the equipment.

#### 2.2.7. Partition Coe fficient Experiments

The partition coe fficient, *Kp*, of the polyfluorenes between the gel-phase DPPG membranes of the TSLs and the aqueous medium was assayed by quantifying fluorescence intensity changes of CPEs in the presence of increasing TSLs concentrations. *Kp* is defined as:

$$K\_p = \frac{n\_L/V\_L}{n\_w/V\_w} \tag{2}$$

where, *ni* and *Vi* correspond with the moles and the volume of phase *i*, respectively. The phase *i* could be either lipidic (*i* = *L*) or aqueous (*i* = *W*). The determination of *Kp* was carried out according to Reference [47]:

$$
\Delta I = \frac{\Delta I\_{\text{max}}[L]}{1/\left(K\_p \gamma\right) + [L]} \tag{3}
$$

where, Δ*I* (Δ*I* = *I* − *I*0) corresponds with the di fference between either fluorescence intensities or emission spectrum areas of the polyfluorenes measured in the presence (*I*) and absence (*I*0) of TSLs, Δ*Imax* = *I*∞ − *I*0 represents the highest value of this di fference once the lowest value is reached (*I*∞) upon increasing the TSLs concentration (*L*), and γ corresponds with the phospholipid molar volume (0.763 <sup>M</sup>−1) [48].

#### 2.2.8. Fluorescence Quenching Experiments

Fluorescence emissions of the CPEs in sodium phosphate bu ffer and integrated in TSLs were studied in the presence and absence of di fferent AQS concentrations. This compound is an electron acceptor which works as a quencher of cationic CPEs, creating static quenching complexes through electrostatic interactions [49,50]. Stern–Volmer analysis was applied to the obtained fluorescence quenching values according to Equation (4):

$$\frac{I\_0}{I} = 1 + K\_{SV}(Q) \tag{4}$$

where, *I* and *I*0 correspond with the steady-state fluorescence intensities in the presence and absence of AQS respectively, and ( *Q*) represents the AQS concentration. The meaning of *KSV* relies on the nature of the quenching process: it could represent the rate of dynamic quenching or the association constant for complex formation (which is the case of AQS and CPEs) [51].

### 2.2.9. Morphological Observation

The morphological observation of the TSLs and multicolor fluorescent nanoparticles was performed by using a transmission electron microscope (TEM) (JEM-1400 Plus, JEOL, Tokyo, Japan), working at 120 kV. A drop of the samples was placed on to 300-mesh copper grips coated with carbon. In order to visualize the vesicles, a drop of lead citrate was also added. Samples were left dry before being placed under the microscope. A Gatan ORIUS camera was employed to record the images.

#### 2.2.10. Cell Imaging Experiments

The human embryonic kidney cell line HEK293 was kindly donated by Dr. Alberto Falcó Gracia (Instituto de Investigación, Desarrollo e Innovación en Biotecnología Sanitaria de Elche, IDiBE, Elche, Spain). Cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) with 10% (*v*/*v*) fetal calfserum (FCS), 2 mM L-glutamine, 100 U/mL penicillin and 100 μg/mL streptomycin and maintained in an incubator with controlled humidity (5% CO2).

For fluorescence microscopy experiments, 96-well plates were used. The final concentration was 10<sup>5</sup> HEK293 cells per mL. Microscopy images were taken in the presence of multicolor fluorescent nanoparticles up to a final CPEs concentration of 0.365 μM, 0.73 μM and 0.18 μM of HTMA-PFP, HTMA-PFNT and HTMA-PFBT, respectively.

Fluorescence microscopy images were captured by using an inverted microscope (Leica DMI 3000B, Leica, Wetzlar, Germany) equipped with a compact light source (Leica EL6000) and a digital camera (Leica DFC3000G). The recording was carried out by using a 63× objective (0.7 magnification) and the filters: DAPI (Ex BP 350/50, Em BP 460/50), FITR (Ex BP 480/40, Em BP 527/30) or DsRed (Ex BP 555/25, Em BP 620/60). Data acquisition was performed manually with Leica Application Suite AF6000 Module Systems, and the image processing was carried out by using the software ImageJ.
