Materials

N-isopropylacrylamide (NIPAM, 99%), OH-terminated Polyethyleneglycolmethacrylate (PEGMA, MW 360, 99%), allylamine (AA, 99%), N, N´-Methylenebisacrylamide (MBA, 99%), sodium dodecyl sulphate (SDS, 99%), ammonium persulfate (APS, 99%), 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC, 99%), N-hydroxysuccinimide (NHS, 99%), 2-(N-morpholino)ethanesulfonic acid (MES, 99%), dimethyl sulfoxide (DMSO, 99.9%) and doxorubicin hydrochloride (DOX, 99.99%) were all acquired from Merck, USA. Folic acid (FA, 99.9%) was acquired from ThermoFisher Scientific, USA. All reagents were used as obtained, without any purification step. Ultrapure water (resistivity 18.2 MΩ cm) was used at all times unless when specified.

Nanoparticles synthesis

Bare thermo-responsive nanoparticles (BNPs) were produced by the free radical polymerization in water of the NIPAM, PEGMA and AA monomers, using MBA as the crosslinker, SDS as the surfactant and APS as the initiator. In more detail, in a 100 mL three-neck round bottom flask, 49 mL of ultrapure water were mixed with the monomers, crosslinker and surfactant, to make a homogeneous solution containing 325 mg of NIPAM, 13.1 mg of MBA, 80 µL of a SDS solution (20% w/v), 30 µL of AA and 25 µL of PEGMA. After solubilization, the solution was placed in an oil bath at 60 °C and purged, for 30 min, with N2 to generate an inert atmosphere. In parallel, 28.5 mg of APS were dissolved in 1 mL of ultrapure water and injected into the previous solution using a needle under N2 protection. The reaction proceeded at 60 °C under slow magnetic stirring (200 rpm). After 4 h, the nanoparticle suspension was allowed to naturally cool to room temperature and was dialyzed (SnakeSkin™ Dialysis Tubing 3.500 MW, Thermo Scientific, USA) against distilled water for 5 days with daily water exchanges. The BNPs were stored in water at room temperature for further use.

Folic acid functionalization

Folic acid conjugation was performed by coupling FA with the free primary amines in the BNPs through the EDC/NHS carbodiimide chemistry. In detail, 107 mg of MES were dissolved in 11 mL of the BNPs suspension and the pH was adjusted to 5.4 ± 0.2 with 1 M NaOH. In parallel, 1 mg of FA, 10 mg of EDC and 10 mg of NHS were dissolved in 1 mL of DMSO and reacted for 1 h to activate the carboxyl groups of FA. Then, 500 µL of the activated FA solution were added dropwise to the BNPs suspension and stirred at 200 rpm for 24 h. Finally, the functionalized nanoparticles (FANPs) were dialyzed as described in the “Nanoparticles synthesis” section, lyophilized at -80 °C for 24 h and stored at 4 °C.

Doxorubicin loading and release

For the loading of DOX into the nanoparticles, a DOX solution (0.6 mg mL− 1) in PBS (pH 7.4) was prepared. After dissolving the drug, lyophilized BNPs and FANPs were dispersed to a concentration of 5 mg mL− 1. The suspensions were then stirred (200 rpm) overnight at room temperature. Unloaded DOX was isolated by centrifugation (14000 rpm for 30 min), the supernatant filtered through a 200 nm syringe filter and the absorbance (λ = 490 nm) was measured using a Synergy MX fluorimeter (BioTek, USA). To determine the concentration, a DOX calibration curve was prepared (R2 = 0.999). Finally, encapsulation efficiency (EE) and drug loading capacity (LC) percentages were determined according to Eqs. 1 and 2, respectively, where Mi represents the initial mass of DOX, Mn represents the mass of non-encapsulated DOX and Mµ represents the initial mass of NPs. DOX loaded nanoparticles (BNPs-D and FANPs-D) were lyophilized (-80 °C) for 24 h in the presence of dextran (5% solution) as a cryoprotectant and stored, protected from light, at 4 °C for further use.

$$\:EE\left(\%\right)=\frac\times\:100$$

(1)

$$\:LC\left(\%\right)=\frac\times\:100$$

(2)

For the drug release studies, lyophilized drug loaded nanoparticles were dispersed in 0.5 mL of PBS (pH 7.4) and placed inside a dialysis bag (SnakeSkin™ Dialysis Tubing 3.500 MW, Thermo Scientific, USA). Sink conditions were maintained by controlling the dialysate volume (minimum 10x times higher than the NPs volume). The bag was then placed in 15 mL of PBS (pH 7.4) at 37 and 40 °C. After each time point, 1 mL of the dialysate was collected and replaced with fresh PBS. Fluorescence (ex. 480 nm and em. 590 nm) was measured using a Synergy MX fluorimeter (BioTek, USA). A calibration curve (R2 = 0.990) was used to quantify the drug release.

Physicochemical characterizationMorphology, size distribution, zeta-potential and thermoresponsiveness

Morphology was assessed by transmission electron microscopy (TEM) in a JEOL JEM-1400 Electron Microscope (JEOL Ltd., Tokyo, Japan) operating at an accelerating voltage of 120 kV. Images were obtained using a CCD digital camera Orious 1100 W (Gatan, USA). Particle size, size distribution and zeta potential measurements were determined through dynamic light scattering (DLS) and electrophoretic mobility in a Zetasizer Nano ZS (Malvern Panalytical, UK) with a scattering angle of 173° and a He-Ne 630 nm wavelength laser. To evaluate the thermoresponsive properties, size was measured at different temperatures through DLS. Temperature was increased in 5 °C steps from 25 to 50 °C with 5 min intervals in between steps for temperature stabilization purposes. Transition temperature values were determined by calculating the inflexion point of the obtained curves. Samples were prepared by dispersing the nanoparticles in ultrapure water and placing them in folded capillary cells (DTS1070, Malvern Panalytical, UK). Each sample was analysed 3 times and each experiment was repeated 3 times (n = 3).

Chemical analysis

Fourier-transform Infrared (FTIR) spectroscopy was employed to identify the chemical groups of each sample. For this, attenuated reflectance mode was used (FTIR-ATR), where 20 µL of a nanoparticle suspension (5 mg mL− 1) in ultrapure water were placed on top of the quartz crystal and allowed to fully dry. Spectra were recorded in a FTIR spectrometer (Perkin-Elmer, USA), with a 4 cm− 1 resolution, with an averaging of 32 scans per sample and within the 4000–400 cm− 1 spectral range.

Micro-Raman spectra were recorded using a Renishaw InVia Qontor spectrometer (Renishaw, UK), equipped with an optical microscope. This study was performed using a 1064 nm laser as excitation. The maximum laser power, on the sample surface, was 100 mW. The microscope is equipped with a 50x long working distance lens and the scattered light was registered in the 3500–100 cm− 1 spectral range. The spectral resolution is better than 2 cm− 1. For this analysis, lyophilized nanoparticles were placed on top of a glass substrate.

UV-Vis spectra were recorded using a UV-Vis spectrometer (Perkin Elmer, USA). For this, the nanoparticles were dispersed in ultrapure water and placed in a quartz cuvette. Scans were recorded in the spectral range of 200 to 600 nm.

Proton nuclear magnetic resonance (1H NMR) measurements were performed with a BRUKER AVANCE III 400 MHz (Bruker Corporation, USA) spectrometer at 25 °C in D2O. Chemical shifts are reported in ppm (δ units) and were referenced to the residual solvent signal.

Biocompatibility studiesCytocompatibility

L929 murine fibroblasts (CCL-1, ATCC) were cultured in α-MEM medium, supplemented with 10% FBS and 1% Pen/Strep, in a humidified atmosphere and incubated at 37 °C and 5% CO2. For cell viability studies, 1 × 104 cells were seeded onto 96-well plates (working volume of 100 µL) and left to adhere for 24 h. BNPs and FANPs were added to the seeded cells at several concentrations in complete medium and incubated for 24, 48 and 72 h. After the time points, medium was removed and replaced with new complete medium containing 10% resazurin (stock solution of 0.1 mg mL− 1) and incubated for 3 h. Finally, the resazurin medium was transferred to 96-well black plates and the fluorescence (ex. 530 nm and em. 590 nm) was measured in a Synergy MX (Biotek, USA). Viability was normalized against the control group and defined as a percentage. Each experiment contained 5 replicates and was repeated 3 times (n = 3). The same experiment was repeated with MDA-MB-468 (HTB-132, ATCC) triple negative breast cancer cells cultured in DMEM medium supplemented with 10% FBS and 1% Pen/Strep, following the same protocol as described above.

Protein adsorption and hemocompatibility

To determine protein adsorption, 1 mg of nanoparticles was incubated with 1 mL of bovine serum albumin solution in PBS (pH 7.4) (BSA, 1 mg mL− 1) for 24 h at 37 °C. After the incubation period, the samples were centrifuged and the supernatant was collected. To determine the BSA concentration a Bradford method was employed by adding 150 µL of Coomassie reagent to 10 µL of sample, mixed for 30 s and allowed to incubate for 5 min at room temperature. Absorbance was read at 595 nm in a multiplate reader (Synergy MX, USA). A BSA standard curve was used to calculate BSA concentrations (R2 = 0.996).

Hemocompatibility was evaluated by the haemolytic and thrombogenic profile of the nanoparticles, under an ethics protocol (Ref. 90/19). For the haemolysis assay, red blood cells (RBCs) were extracted from human buffy coats by centrifugation at 400 g for 30 min. Following centrifugation, the upper layer was discarded and the pellet was washed 3x with PBS (pH 7.4). The nanoparticles were incubated with isolated RBCs (2 × 108 cells mL− 1) in 96-well suspension plates for 3 h at 37 °C. Subsequently, the plates were centrifuged at 4000 rpm for 15 min. 100 µL of the supernatant were transferred to 96-well plates and the absorbance was measured at 380, 415 and 450 nm, using a microplate reader spectrophotometer Synergy MX (Biotek, USA). The amount of released haemoglobin (RH) was calculated following Eq. 3 where A415, A380, and A450 represent the absorbance values of the samples at 415 nm, 380 nm, and 450 nm, respectively. E is the molar absorptivity of oxyhaemoglobin at 415 nm. Hemolysis was calculated according to Eq. 4, where RH represents the total release of haemoglobin in Triton X-100 1%. The test was performed 3 individual times (n = 3) with 5 replicates within each assay.

$$\:RH\left(mg\:^\right)=\frac_-(_+_)\times\:1000}$$

(3)

$$\:Haemolysis\left(\%\right)=\frac$$

(4)

The effect of the nanoparticles on plasma coagulation was evaluated by the plasma coagulation kinetics assay, using human plasma under an ethics protocol (Ref. 90/19). For this, silica nanoparticles and PBS were used as positive and negative controls, respectively. 50 µL of a nanoparticle suspension (4 mg mL− 1), or the controls, were mixed with 450 µL of human plasma and incubated for 30 min at 37 °C. After incubation, 50 µL of the nanoparticle + plasma mixture were placed in 96-well suspension plates, to which 50 µL of a CaCl2 solution (5 mM) were added. The absorbance was measured at 405 nm for 180 min, with 1 measurement per minute. The coagulation time was determined at the inflexion point of the obtained curves.

Anti-cancer activity in 2D modelsCancer cell viability studies

MDA-MB-468 (HTB-132, ATCC) triple negative breast cancer cells were cultured in DMEM medium containing 10% FBS and 1% Pen/Strep, in a humidified incubator at 37 °C and 5% CO2. For cell viability studies, 1 × 104 cells were seeded in 96-well plates (working volume of 100 µL) and allowed to adhere for 24 h. BNPs-D and FANPs-D were added to the seeded cells at several concentrations in complete medium and incubated for 24, 48 and 72 h. After the time points, the resazurin method was employed as in the “Cytocompatibility” section. Viability was normalized against the control group and defined as a percentage. Each experiment contained 5 replicates and was repeated 3 times (n = 3).

Folate receptor competition assay

MDA-MB-468 (HTB-132, ATCC) were grown and seeded in 96-well plates as in the “Cancer cell viability studies” section. Before the addition of the nanoparticles, some cell conditions were incubated with a FA solution (5.0 mM) in complete medium for 1 h to saturate the FOLR1 receptors. After incubation, BNPs-D and FANPs-D were added at a DOX concentration of 100 µM and incubated for 24 and 72 h. Finally, the resazurin method was employed as in the “Cytocompatibility” section. Viability was normalized against the control group and defined as a percentage. Each experiment contained 5 replicates and was repeated 3 times (n = 3).

Anti-cancer activity in 3D modelsSpheroids optimization

3D cell constructs (spheroids) were developed using commercially available micromolds (3D Petri Dish®, MicroTissues, USA), following the manufacturer’s instructions. Culture was performed in low-adhesion 12-well plates and the working volume was set to 1.5 mL. MDA-MB-468 cells were cultured as in the “Cancer cell viability studies” section, trypsinized and seeded in the agarose micromolds at different cell densities per spheroid (1 × 103, 2.5 × 103 and 5 × 103). Medium was replaced every 2 days. At different timepoints (1, 4 and 7 days), cells were imaged by brightfield microscopy (ZOE™ Fluorescent Cell Imager, Bio-Rad Laboratories, USA). Size was evaluated through the ImageJ software version 1.8.0 (NIH, USA), by taking 4 diameter measurements in 3 randomly selected spheroids. Viability was assessed by the resazurin assay by replacing the growing medium with fresh medium containing 20% resazurin, followed by a 2 h incubation period. Medium was transferred to black 96-well plates and the signal fluorescence was measured (ex.530 nm, em.590 nm) in a plate reader (SynergyMX, BioTek, USA). To complement the viability studies and understand the structure of the spheroid, a live-dead assay was performed. After the timepoints, each mold was washed with PBS and incubated with Calcein-AM (1:1000 dilution) for 15 min in complete medium, followed by incubation with a solution of propidium iodide in PBS (25 µg mL− 1) for 15 min. Finally, the molds were washed with PBS and analysed by fluorescence (Calcein: λ = 488 nm; Propidium iodide: λ = 561 nm) microscopy using a SP5 confocal microscope (Leica Microsystems, Germany) and image analysis was performed with the LAS AF Lite software (Leica Microsystems, Germany) and with the ImageJ software version 1.8.0 (NIH, USA). Images were created by combining z-stacks.

NPs anti-cancer activity in 3D cultures

MDA-MB-468 spheroids were produced as described in the “Spheroids optimization” section. At day 4, medium was replaced with medium containing free drug or FANPs-D at several concentrations for 72 h at 37 and 40 °C. After 72 h, the spheroids were washed with PBS and incubated for 2 h with complete medium containing 20% resazurin. Medium was transferred to black 96-well plates and the signal fluorescence was measured (ex. 530 nm, em. 590 nm) in a plate reader (SynergyMX, BioTek, USA). Each experiment contained 4 replicates and was repeated 3 times (n = 3).

Doxorubicin penetration evaluation

Spheroids with an initial cell seeding density of 2.5 × 103 cells per spheroid were produced as presented in the “Spheroids optimization” section. At day 4 of culture, the spheroids were exposed to the free drug or to FANPs-D at 37 and 40 °C. After 6 h, spheroids were washed 3x with PBS and fixed for 15 min with a 4% paraformaldehyde solution. After fixation, the molds were washed with PBS and analysed by fluorescence microscopy, taking advantage of DOX fluorescence (ex. 480 nm and em. 590 nm), using a SP5 confocal microscope (Leica Microsystems, Germany) and image analysis was performed with the LAS AF Lite software (Leica Microsystems, Germany) and with the ImageJ software version 1.8.0 (NIH, USA). Images were created by combining z-stacks.

Ethics statement

Blood components were obtained from surplus buffy coats from healthy blood donors, kindly provided by the Immunohemotherapy Department of Centro Hospitalar Universitário São João (CHUSJ), Porto, Portugal. Procedures were approved by the Centro Hospitalar Universitário São João Ethics Committee (protocol 90/19). Blood donors provided informed written consent that the by-products of their blood collections could be used for research purposes.

Statistical analysis

Statistical analysis was performed with the GraphPad Prism 8 software (GraphPad, USA). Data are represented as mean ± standard deviation (SD). Statistical analysis was performed using the analysis of variance One-way ANOVA or Two-way ANOVA, followed by Tukey’s multiple comparison test. Statistical significance was considered when p < 0.05 and represented with * for p < 0.05, ** for p < 0.01, *** for p < 0.001 and **** for p < 0.0001.